ms 1 .,.~ _, i_, This is to certify that the thesis entitled INHIBITION OF N-NITROSAMINE FORMATION IN MODEL AND MEAT SYSTEMS presented by William George Ikins has been accepted towards fulfillment of the requirements for M.S. degree in FOOd SCIenQe' [ff/9M7 Major #fessorv £67 29, mm (I / 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution In»K IlfliillllllfllmflilWill 3 1293 10763 675 MSU RETURNING MATERIALS: Place in book drop to LIBRARJES remove this checkout from .—:—. your record. FINES W‘III be charged if book is returned after the date stamped below. J; a; '2 9 "15:13 2‘! 1 I p 1 AUG 2 2093 A R ,g q.._' ,. 5,2,1 , T i _, 'I II I ' . A $1 \ I . “AA-3A9 *2 ., . a . ..;9 0 ,3?» « “’KZI’L‘E‘IR ’1‘” {wwwln ~— 35 '- " --2 {AK 4! I") g; 4‘ £112; ‘ $§§§g urn-M”, A ‘I '1’... 3) 13):; ‘- I12} I 3% ‘ I 3 INHIBITION OF N-NITROSAMINE FORMATION IN MODEL AND MEAT SYSTEMS By William George Ikins A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1982 ABSTRACT INHIBITION OF N-NITROSAMINE FORMATION IN MODEL AND MEAT SYSTEMS By William George Ikins The effectiveness of several compounds as potential blocking agents for the N-nitrosation reaction was investi- gated using model systems. Ascorbic acid effectively blocked N-nitrosopyrrolidine (NPYR) formation in an aqueous model system, but accelerated the reaction in a lipid- aqueous model system. Lipophilic derivatives of ascorbic acid were effective in the two phase model system at 25°C, but enhanced NPYR formation at 80°C. The flavonoids, rutin, quercetin dihydrate, and genistein, enhanced NPYR formation at low concentrations but exerted an increasing inhibitory effect with increasing concentration. These compounds were not effective in the two phase model system. Unlike the other compounds, the antioxidant ethoxyquin was a more effective blocking agent at 80°C than 25°C in the two phase model system. Soy flour effectively blocked NPYR formation in an aqueous model system. When included in a frankfurter emulsion spiked with a secondary amine, however, soy flour accelerated the formation of N-nitrosodimethylamine (NDMA) in heated and unheated frankfurters. Dedicated to my family, Dr. Phillip and Marion Ikins and Doctors Fredrick and Laura Stern and their children David, Georgette and Lance Stern 1'1 ACKNOWLEDGEMENTS The author wishes to express his sincere thanks and deepest gratitude to Dr. J.I. Gray, whose patience, guidance, and encouragement made the completion of this thesis possible. Genuine appreciation is extended to the members of the research guidance committee, Dr. L.E. Dawson, Dr. A.M. Pearson, and Dr. J.F. Price of the Department of Food Science and Human Nutrition, and Dr. B.R. Harte of the Department of Packaging for their critical review of this thesis. The author is also indebted to several individuals for their friendship and technical assistance in this research, including Arun Mandagere, Sue Cuppett, and Man Lai Lee. Finally, the author is especially grateful for the unlimited support provided by Nancy J. Bates and Booker. TABLE OF CONTENTS LIST OF TABLES. LIST OF FIGURES INTRODUCTION. LITERATURE REVIEW Chemistry of Formation. N-nitrosamine reactions . . . Kinetics of N- nitrosation . . . . Occurrence of N- Nitrosamine in Foods. . Factors Influencing N- Nitrosamine Formation in Foods . . Inhibitors of N- Nitrosamine Formation Ascorbic acid . . Ascorbic acid derivatives Alpha tocopherol. Phenols Sulfur compounds. . . Antioxidant activity of soybeans. EXPERIMENTAL. Materials Methods Aqueous model systems . . Model systems containing potential block- ing agents. . Model systems containing defatted soy flour . . . Model systems containing methanol and residue of defatted soy flour . . Lipid model systems . . . . . . . . . . . Ground adipose tissue system. Corn oil- buffer model system. Frankfurter study . . . . Analytical techniques . GC analysis of N- nitrosamines TEA analysis of N- nitrosamines. . . HPLC analysis of soybean isoflavones. iv Page vi viii RESULTS AND DISCUSSION. Aqueous Model Systems . Model systems containing potential blocking agents. . Model system containing defatted soy rour. Model system containing alcoholic extracts or residues from soy flour . Lipid Model Systems . Potential blocking agents in ground adipose tissue. . Potential blocking agents in a two phase model system. . . . . . Frankfurter Study . SUMMARY AND CONCLUSIONS . PROPOSALS FOR FURTHER RESEARCH. BIBLIOGRAPHY. Page 55 55 55 69 73 77 77 BI 87 92 '95 '96 Table TO LIST OF TABLES Effects of ascorbic and dehydroascorbic acid on N- nitrosamine formation in an aqueous model system (pH 3. 5) containing PYR (l mM) and NaNOz (2 mM). . . . . . . . . . . Effect of quercetin dihydrate on N-nitrosamine formation in an aqueous model system containing PYR (1 mM) and NaNOz (2 mM) . . Effect of rutin on N- nitrosamine formation in an aqueous model system containing PYR (l mM) and NaNOz (2 mM). . . . . . . . . . . . . Effect of genistein on N-nitrosamine formation in an aqueous model system at pH 3.5. Effect of Biochanin A on N-nitrosamine formation in an aqueous model system of H 3.5 containing PYR (.02 mM) and NaNOz (.04 mMI . . . Effect of ethoxyquin on N- nitrosamine formation in an aqueous model system containing PYR (l mM) and NaNOz (2 mM). . . . . . . . . . The effect of soy flour on the formation of N- nitrosamines in an aqueous model system at pH 3.5 containing PYR (l mM) and various concentra- tions of nitrite. . . . . . . . . . . . . . Quantity of aglycone isoflavone, glycoside iso- flavone and major cinnamic acids in 20 g of defatted soy flour. . The effect of a methanol extract and residue from 20 g of soy flour in an aqueous model system at pH 3.5. . . . . . . . . The effect of various potential blocking agents on NPYR formation in a ground adipose tissue model system. . . . . . . . . . . . vi Page 57 6O 64 66 66 68 7O 71 75 78 Table ll 12 13 14 The effect of various potential blocking agents on NPYR formation in a ground adipose tissue model system. The effect of various blocking agents on NPYR formation in a two phase model system of buffer (pH 5.5) and corn oil . . . . . . . . Nitrite contents of frankfurters prepared with varying levels of soy flour . . . Effect of increasing levels of soy flour on the N-nitrosamine content of frankfurters Page 80 82 89 9O LIST OF FIGURES Figure Page l Reaction of ascorbic acid with nitrite. . . . . . I9 2 Reaction of cxtocopherol with nitrite . . . . . . 27 3 Reaction of phenols with nitrite. . . . . . . . . 31 4 Structure of the major isoflavone glycosides. . . 40 5 Proposed mechanism of catalysis of N-nitrosamine formation by 1,3 dihydroxyphenols (Walker et 11., 1982) EN 6 Structures of potential blocking agents . . . . .° 52 7 Postulated free radical mechanism of NPYR forma- tion (Bharucha et 31., I982). . . . . . . . . . . 85 viii INTRODUCTION A severe outbreak of liver disease in mink and sheep in Norway in the early 1960's was attributed to the herring meal with which the animals were fed (Kappang, I964). The causative agent was identified as N-nitrosodimethylamine (NDMA), presumably formed by the interaction of high levels of sodium nitrite added to the meal as a preservative and amines naturally present in fish (Ender et 11., l964). Scientists quickly became aware that carcinogenic N-nitro- samines could be formed in human food, particularly those preserved with nitrite. Subsequently, a variety of foods have been found to contain significant quantities of N-nitro- samines (Gray, 198l). The detection of N-nitrosamines in food products had a most profound effect on the cured meat industry, which is heavily dependent on nitrite for its existence. In order to minimize N-nitrosamine formation in cured meats, the levels of nitrite added have been gradually lowered, but are still sufficient to retain a safety margin against the threat of botulism. Another approach to minimize N-nitrosamine formation in cured meats has been the addition of compounds (blocking agents) that can effectively inhibit the N-nitrosation of amines. It is essential that the blocking agent be already approved for food use by the Food and Drug Administration or be of natural origin because of the long expensive safety testing required of novel synthetic blocking agents. Ascorbic acid is approved for food use and has been reported to be an effective blocking agent of N-nitrosamine formation in frankfurters (Fiddler e; 11., 1973). Sby flour is permitted in emulsified meat products by U.S.D.A. regulations at levels up to 3.5% (Sofos et 31., 1977). However, frankfurters have a potentiai for inclusion of higher levels of soy products. Sofos et’gl. (1977) incorporated textured soy protein into frankfurters emul- sions at levels up to 30% with no significant loss of sensory evaluation scores. The substitution of plant protein for animal protein is advantageous because of the reduced cost of the frankfurter. It has also been reported that antioxidants such ascrtocopherol and ethoxyquin have been used successfully as blocking agents in model systems and cured meat products (Coleman, 1978; Fiddler et 11., 1978). Soyflour contains significant concentrations of the phenolic cinnamic acids and glycosidic isoflavones, compounds which have been demonstrated to possess appreci- able antioxidant activity (Pratt and Birac, 1979). Therefore, the primary objective of this study was to determine if soy flour could function as an inhibitor of the N-nitrosation reaction in model and cured meat systems. A secondary objective was to examine which constituents of soy flour were responsible for the blocking effect. LITERATURE REVIEW One of the major concerns of the food industry over the last fifteen years has been the presence of N-nitrosa- mines in consumer products. This concern has resulted in a vast amount of research into all aspects of this problem. Approximately 25 years ago, it was discovered that low concentrations of N-nitrosodimethylamine (NDMA) caused liver cancer in rats (Magee and Barnes, 1956). Since then, more than 120 N-nitroso compounds have been tested, 80% of which have shown carcinogenic activity (Magee et 11., 1976). This activity has been demonstrated in twelve species of animals, including monkeys. The majority of the research on N-nitrosamines has focused on cured meats, particularly bacon. These products are likely media for formation of these compounds because of the abundance of amines in a meat system, and the addition of nitrite, which gives the product its cured meat characteristics and protection against botulism. Research into minimum concentrations of nitrite necessary in cured meats as well as more careful monitoring of the levels of nitrite added has resulted in a lowering of the N-nitrosamine levels in bacon over the last decade (Gray, 1981). However, cured meats have not been the only product to encounter this problem. The discovery of NDMA in beer forced brewers in North America to modify their malting procedures (Havery gt 31., 1981). In addition, N-nitrosa- mines have been found in non-food items, such as cosmetics, cutting fluids and rubber products (Preussman et al., 1981). A more difficUlt problem to research has been the N-nitrosation of amines 1 vivo, within the favorable environment of the stomach and intestines (Lijinski et.gl., 1970). In this review, the chemistry of formation of N- nitrosamines will be examined, including a discussion of the N-nitrosation reactions and the kinetics of these mechanisms. Additionally, the occurrence of N-nitrosamines and the factors affecting their formation in food products will be reviewed. The main thrust of this review deals with compounds capable of inhibiting N-nitrosamine forma- tion (blocking agents), with particular emphasis on the phenolic inhibitors. Chemistry of Formation N-Nitrosamines are stable compounds which are formed principally from the reaction of secondary amines with a N-nitrosating species. R R ' RI>NH + HN02——R1>‘i-N=0 + H20 (1) R1 is an alkyl group, while R may be any one of a large number of functional groups. N-Nitrosamines can also be formed from primary amines, tertiary amines, and quaternary ammonium compounds (Fiddler e} 11., 1972). The carcinogenic activity of N-nitrosamines depends upon the structure of the compound (Wishnok et 31., 1978). It appears necessary for the N-nitrosamine to possess an a hydrogen that can be enzymatically hydroxylated to begin the modification of the N-nitrosamine. After several steps, either a diazonium ion or a diazoalkane is produced which can alkylate the nucleophilic sites on deoxyribonu- cleic acid (DNA), ribonucleic acid (RNA), or proteins, thus producing the carcinogenic effect (Magee and Barnes, 1967). N-Nitrosamine Reactions Primary amines can undergo conversion to a secondary amine and ultimately to a N-nitrosamine under cold acidic conditions (Ridd, 1961). The rapid reaction proceeds through an unstable primary N-nitrosamine to a diazonium intermediate, which then can react with the primary amine starting material to form a secondary amine. ””2 + NOZ'HI—q-R-N-Nzo r R-NEN (2) H 10 amine 1o N-nitrosamine diazonium ion R NOZ‘HI -N2 + R \ \ R/ R/ 2 At this low pH, nitrous acid (HONO) or nitrous acidium ion (HZNOZT) become the important N-nitrosating species. In order for the secondary amine to be N-nitrosated, it must be in the unprotonated form. Since the above reaction occurs in a very acid environment, a low proportion of the secondary amines will be unprotonated, and consequently a low yield of N-nitrosamines will result. Primary diamines with chains of four or five carbons, such as putrescine, can cyclize to form secondary amines and ultimately N- nitrosamines under high temperature conditions or with long reaction time (Warthesen e; 11., 1975). Secondary amines are N-nitrosated at a maximum rate in an aqueous environment at a pH of 3.4. Under these mildly acidic conditions, nitrous anhydride (N203) becomes the principal N-nitrosating species. 2111102211203 + H20 (3) RZNH + N203—-R2NN=O+HN02 (4) 2° amine Tertiary amines have generally been regarded as inert to N-nitrosation because of the high temperature dealkyla- tion required to obtain a secondary amine (Smith and Loeppky, 1967). The tertiary amine is N—nitrosated to a N-nitrosammonium ion, which then undergoes cis elimination of the nitroxyl ion to form an iminium ion. This ion is then hydroxylated to give an aldehyde or ketone and a secondary amine, R1 ll,1 + R-N—CHZRZ + N203 #R-TI-CHZRZ—I-HNO (5) N H o 3°amine nitroxyl ion + R-N-N=0 —-———R-NH + RZC—H—-—R-N=CH2R2 N203 + 2° amine aldehyde iminium ion which can then be N-nitrosated to the corresponding N-nitrosamine. Quaternary ammonium compounds react even more slowly than tertiary amines (Fiddler 33 31., 1972). An initial dealkylation must occur before any N-nitrosating agent can become involved, as in reaction (5). Besides the N-nitrosating species already mentioned, nitrogen oxides such as nitrogen dioxide (N02), dinitrogen tetraoxide (N204), and nitric oxide (NO) have also been implicated in the formation of N-nitrosamines. In order for NO to act as a N-nitrosating agent, it must either react with an amine under anaerobic conditions in the presence of certain metal salts, or be oxidized to N02 (Douglass 31 31., 1978). Gaseous N204 exists in equilibrium with its N02 N204:2N02 (6) constituent (Hisatsune, 1961). N-Nitrosation by N204 yields a mixture of N-nitro and N-nitroso amino compounds (Challis and Kyrtopoulos, 1978). These reactions will RZNH + N204—u-R2NN0 + HNO3 (7) RNH+NO—DR NNO 2 24 2 ZIHNO 2 (8) occur in a gas phase, an organic or lipophilic environment, and in a neutral or alkaline aqueous solution. Kinetics of N-Nitrosation The N-nitrosation of a secondary amine under mildly acidic conditions proceeds via the mechanism depicted in reactions 9, 10 and 11 (Mirvish, 1975). The reaction kinetics can be expressed in one of two ways. In equation 12, the rate of N-nitrosation is proportional to the ._a 2HN02‘_N203 + H20 (9) RNH++H0‘—RNH+H0+ (10) 2 2 2 ‘—=' 2 3 N203 + RZNH—I-RZNNO + H1102 (11) - 2 Rate - k1 (RZNH) (HNOZ) (12) Rate = k2 (amine) (nitrite)2 (l3) 10 concentration of nonionized amine, since only the unproto- nated secondary amine can be N-nitrosated. The unprotonated amine exists in equilibrium with its conjugate acid, as shown in reaction 10. The rate is also proportional to the (31203), and thus to 01110232. Although k] will be indepen- dent of pH, the concentration of HNO2 and RZNH must be calculated at each pH. Equation 13 offers the convenience of using the total concentration of amine and nitrite regardless of their ionic state. However, k2 will vary with pH. In general, the reaction rates are dependent on pH, the basicity and concentration of the amino substrate, the nitrite ion concentration, and the presence of catalytic anions. The effect of low pH on the reaction kinetics has already been discussed. As the pH is raised above the maximal 3.4, N-nitrosation steadily decreases and stops altogether at a pH of 6.0. However, in the presence of formaldehyde, the pH over which N-nitrosation can occur is extended well into the alkaline range (Keefer and Roller, 1973). The nature of the amine will influence the reaction rate, in that as the basicity or pKa of the amine decreases, the ease of N-nitrosation will increase. In a N-nitrosation reaction catalyzed by an anion, nitrite must first be converted to nitrous acid in an acid catalyzed reaction. The NO group is then passed on to a catalytic anion, represented by the symbol Y' in the following reactions. 11 The nitrite ion (NOZ') will act as the nucleophile (and +- ‘__ + 111102 + H30__, H20 -— N0 (14) H20+— N0 + Y‘ _,""' Y—NO + H20 (15) RNH + YNO ilfll—RZNNO + HY (16) thus N203 as the N-nitrosating agent) under the appropriate conditions or in the absence of other stronger nucleophiles, such as thiocyanate (NCS'), bromide (Br'), iodide (I'), or chloride (Cl'). In an environment of low pH (<2.5), or when the concentration of thiocyanate or halide is high and that of nitrite is low, the thiocyanate and halide cata- lyzed mechanism can dominate (Fan and Tannenbaum, 1973). This could have important implications for cigarette smokers whose thiocyanate levels in saliva and gastric juices are higher than those of nonsmokers (Lane and Bailey, 1973). However, experiments involving rats fed NaNCS, nitrite and amines have not demonstrated in vivo catalysis (Lane and Bailey, 1973). There are, of course, many exceptions to these basic kinetic equations. The N-nitrosation of N-methylaniline is so fast that the formation of N203 becomes rate limiting, and thus the kinetics are described by reaction 17. Should the N-nitrosation of this amine occur at pH 1, however, the 12 (17) reaction rate is more accurately described by equation 12, because of the low concentration of nonionized amines. Aminopyrine is a tertiary amine that is rapidly N-nitro- sated to form NDMA, and its kinetics are also best described by equation 17. The N-nitrosation of amino acids is quite rapid, but complex in terms of reaction kinetics. The difficulty lies in the fact that the protonation of the carboxyl group affects the basicity of the amino group (Mirvish 33 31., 1973). Temperature has a profound effect on the N-nitrosation reaction rate. Foreman and Goodhead (1975) reported that for every 10°C rise in temperature, the reaction rate ' doubled. Freezing does not stop N-nitrosation either. Fan and Tannenbaum (1973b) demonstrated an enhancement of the N-nitrosation of morpholine in frozen buffers and milk, which was attributed to the concentration of the reactants under frozen conditions. Occurrence of N-Nitrosamines in Foods Trace amounts of N-nitrosamines have been reported in a variety of foods. Of these food products, cooked bacon, nitrite or nitrate-treated smoked fish, and salted or dried ocean fish appear to yield the most consistently high concentrations of N-nitrosamines (Sen, 1980). These 13 compounds have been sporadically found in certain frank- furters, sausages, and salami, all made with premixed spices. However, the N-nitrosamines were discovered to be generated not from the processing of the meat, but in the spice premix where nitrite was free to react with the amines in the spices (Sen 31__1,, 1974). The separate packaging of nitrite and spices greatly reduced N-nitrosamine concen- trations in these products (Sen and McKinley, 1974). The presence of N-nitrosamines in cooked bacon has been the major concern of the cured meat industry, and has been widely investigated. Although as many as four dif- ferent N-nitrosamines have been detected in cooked bacon (Crosby 31 31., 1972), N-nitrosopyrrolidine (NPYR) and DMNA have consistently been found (Gough and Walters, 1976). Free proline is the most probable precursor of NPYR, and is present in substantial amounts (20-80 mg/kg) in connective tissue of pork bellies (Gray and Collins, 1977). Although there is a great deal of controversy about the pathway of NPYR formation, the most probable mechanism begins with the N-nitrosation of proline to form the N-nitrosoproline intermediate during the high temperature frying of bacon, followed by decarboxylation to form NPYR. Bharucha 33_31. (1979) have suggested that the formation of NPYR in bacon occurs almost entirely in the fat phase after the bulk of the water is removed, and therefore by a free radical rather than an ionic mechanism. 14 Cured smoked fish and salted dried marine fish are a dietary staple for people in certain parts of the world. Significant quantities of N-nitrosamines have been encoun- tered in fish, ranging as high as 100 mg/kg of NDMA in a nitrite-treated herring meal (Ender 31 31., 1964). In comparison to fresh water fish, marine fish are much more likely to contain high levels of N-nitrosamines because of their higher amine content (Sen, 1980). Nitrite or nitrate is added as a cure ingredient or can be present as an impurity in the crude salt (Fong and Chan, 1976). The potent carcinogen NDMA is the most frequently found N- nitrosamine in marine fish (Fazio 31 31., 1971; Fong and Chan, 1973). Other foods in which N-nitrosamines have been detected include various cheeses, mushrooms, a solanaceous fruit, and alcoholic drinks (Crosby 31_31., 1972; Ender and Ceh, 1967; DuPlessis 31 31., 1969; Bogovski 31 31,, 1974). In general, it can be said that these products present little threat since N-nitrosamines were found in low concentrations and in a small percentage of the samples (Sen, 1980). factorslnfluencing N-Nitrosamine Formation in Foods The factors influencing the formation of NPYR in cooked bacon have been well documented (Gray, 1981), and it is likely that the formation of otherN-nitrosamines are affected by these same variables. These factors include 15 the method of cooking, cooking temperature and time, nitrite concentration, preprocessing procedures, presence of lipophilic inhibitors, ascorbate concentration, sodium chloride concentration, and possibly smoking. Several methods of cooking bacon were examined by Pensabene 31 31. (1974) and it was concluded that the frying temperature was more influential than time on NPYR formation. Bacon, from one belly produced no NPYR when fried for 105 minutes at 99°C, while samples from the same belly, fried to the same "doneness" at 204°C for 4 minutes, produced 17 ug/kg of NPYR. Baking produced the highest single sample yield of 35 ug/kg, while microwave cooking yielded essentially no NPYR. Bharucha 31 31. (1979) obtained a reduced yield of NPYR in bacon lean while grilling the meat as opposed to pan frying. These authors attributed this reduction to lower frying temperatures that result when the bacon fat is allowed to mni out of the heated area. It should be noted that about 50% of the NPYR and 70% of the NDMA are released as vapor during the frying of bacon. 0f the remaining N-nitrosamines, approximately two thirds are retained in the cook-out fat and one third in the lean (Sen, 1980). From the kinetics of N-nitrosation, it can be seen that the reaction rate is directly proportional to the square of the nitrite concentration. In light of this relationship, the levels of nitrite permitted in the 16 processing of meat have undergone a great deal of scrutiny. Dudley (1979) reported that it is the lowest residual nitrite concentration rather than the lowest initial nitrite concentration that determines the probability of N-nitrosamine formation during frying. The length of storage of a pork belly prior to proces- sing has an important influence on the final nitrosamine content of the fried bacon. Gray and Collins (1977) repor- ted that the proline concentrations increased approximately 50% in the lean tissue and 90% in the adipose tissue of green pork bellies over a one week storage period at 20°C. Pensabene 31 31. (1980) concluded that bacon made from fresh bellies produced significantly less NPYR than that made from bellies that had either been stored for 1 week in a refrigerator or frozen for 3 months and then thawed before using. Ascorbic acid has long been used to accelerate color de- velopment in cured meats. It also appears that ascorbate can enhance the antibotulinal properties of nitrite (Tompkins 31 31., 1978). In addition, ascorbic acid and several lipophilic blocking agents can inhibit the formation of N- nitrosamines. This topic will be discussed in the next section of this review. Because sodium chloride has traditionally been an integral part of cure mixtures, its effect on N-nitrosation has been widely studied. Hildrum 31 31. (1975) reported 17 that sodium chloride has an accelerating effect on the N-nitrosation of proline at pH 0.5, a mild inhibitory effect at pH 2.5, and a moderately inhibitory influence at pH 4.0 and 5.5. The authors theorized that the catalyst, nitrosyl chloride, was the dominant N-nitrosating species at the lowest pH. As the pH increases, nitrous anhydride becomes more prevalent as the N-nitrosating species, and the concentration of nitrosyl chloride diminishes. Thus, at the mildly acidic pH of meat, chloride ions would be expected to exert a mildly inhibitory effect on the N- nitrosation reactions. The effect of smoking on the N-nitrosamine content of bacon is not a profound one. Bharucha 31 31. (1980) reported that unsmoked bacon samples generally tended to contain more N-nitrosamines, presumably because of their higher nitrite content at the time of frying. Sink and Hsu (1977) demonstrated a lowering of residual nitrite in a liquid smoke dip process for frankfurters when the pH is also lowered. The lowering of residual nitrite levels in smoked bacon is the result of the pH decrease and the direct C-nitrosation of phenolic compounds which are deposited on the surface of the bellies during smoking (Knowles, 1974). However, C-nitrosophenols can act as powerful catalysts to N-nitrosamine formation as will be discussed in the follow- ing section of this review. 18 Inhibitors of N-Nitrosamine Formation Any compound that can successfully compete with a secondary amine for a N-nitrosamine species would reduce the possibility of N-nitrosamine formation (Gray and Dugan, 1975). These compounds are called blocking agents and generally function by reducing the N-nitrosating species to a nonnitrosating oxide of nitrogen of lower oxidation state (Mergens and Newmark, 1980). In the following section, the effects of selected compounds investigated as N-nitrosamine blocking agents will be discussed. Ascorbic Acid Ascorbic acid was first discussed as a N-nitrosamine blocking agent by Mirvish 31 a]. (1972). Their major concern was the N-nitrosation 13.1113 of drugs such as piperazine, which is used for killing pinworms in children. The mecha- nism by which ascorbate competes with amines for N-nitrosating species is shown in Figure 1. The effectiveness of ascor- bate is primarily dependent on the amine with which it is competing, as well as the pH of the environment, which determines the N-nitrosating and ascorbic acid species. As a result of its greater neucleOphilic activity, the ascor- bate anion is 230 times more reactive than ascorbic acid, and is present to the greatest extent in the pH range cf 3 to 5 (Dahn 31 31,, 1960). .muwcpw: gum: uwom peacoUmm wo cowuummm .P mczmru Eo< 0333.06.52.00 .Eo< o_n.ooa< o o . o o : gouzu+oz~ + :ozouxoo: I «02.3 + :ozouxoo: o o. :o . o: 20 Using five different amines of varying basicity, Mirvish 31 31. (1972) evaluated the effectiveness of ascor- bate in an aqueous system over a range of pH values. Ascorbate was effective in blocking the N-nitrosation of morpholine and piperazine, two amines which do not react rapidly. N-Methylanaline, on the other hand, reacted with nitrite at a similar rate as ascorbate, and as a result, only 60% inhibition of the N-nitrosation reaction was achieved. The N-nitrosation of dimethylamine (DMA) was effectively inhibited by ascorbate once conditions were adjusted to obtain greater than 1% yield of the N-nitrosa- mines. Oxytetracycline, a tertiary amine that is a precur- sor of NDMA, was blocked even more efficiently than N- methylanaline by ascorbate under similar conditions. Sen 31 31. (1974) confirmed that ascorbate blocked the formation of mononitrosopiperazine from a piperazine containing drug, although they were not able to achieve as high a percentage of inhibition as Mirvish 31 a_1_. (1972). However, ascorbate accelerated the formation of the more carcinogenic dinitrosopiperazine from the same drug, even though free piperazine formed little dinitrosopiperazine in the presence of ascorbic acid. Archer 31 31. (1975) reported that complete inhibition of N-nitrosomorpholine formation can be achieved using an ascorbate/nitrite ratio of 2:1 at pH 4.0. When air was bubbled through the reaction mixture, the ascorbate 21 concentration required for complete inhibition of the nitrosation of morpholine was increased. The researchers theorized the air oxidized the ascorbate and made it A unavailable for reaction with nitrite. Gray and Dugan (1975) investigated the influence of ascorbic acid on the nitrosation of DMA in aqueous and low moisture model systems. An ascorbate/nitrite ratio of greater than 2:1 was deemed necessary to totally block the formation of NDMA. These authors further subjected the low moisture carboxymethylcellulose system containing added proline to heat stress (175°C for 45 minutes). Ascorbic acid was again found to effect greater than 95% inhibition of NPYR formation. Kawabata 31 31. (1974) noted that at pH 6.0, at least a 2:1 ratio of ascorbate/nitrite was required before significant inhibition of NDMA formation occurred. Ascorbic acid concentrations equivalent to one tenth the nitrite concentration or less caused acceleration of N-nitrosamine formation at pH 6.0, regardless of tempera- ture. Again, maximum inhibition of NDMA formation occurred at the optimum pH of 3.6. In agreement with the findings of Mirvish 31 31, (1972), Fan and Tannenbaum (1973) demonstrated that when the ascorbate/nitrite ratio was greater than 2, total inhibition of N-nitrosomorpholine formation was achieved in an aqueous system. The authors reported that as the ratio of ascorbate/nitrite was increased, the residual 22 nitrite decreased in a linear fashion. With no ascorbate present, the nitrite concentration changed very little as the pH was slowly decreased from 5 to 2. In the presence of ascorbate, however, more than half the nitrite was lost as the pH was lowered. This was probably due to the vola- tilization of nitric oxide, a product of the reduction of nitrite by ascorbic acid. To obtain a better understanding of how N-nitrosamines are formed in food products like bacon, Mottram and Patter- son (1977) employed a two phase model system of buffer and corn oil or benzene. Sodium ascorbate was discovered to greatly increase the yield of NPYR and N-nitrosodipropyla- mine. The authors theorized that when nitrites are reduced in a purely aqueous system, the nitrogen oxides volatilize from solution or are kept in a non-nitrosating form (NO) by the reducing agent. In a two phase system, nitrogen oxides are free to migrate into the nonpolar phase away from the polar ascorbic acid, and N-nitrosate amines there. In order to evaluate the effect of ascorbate on N- nitrosamine formation in a meat system, Mottram et a1. (1975) used pork slices spiked with DMA to reduce varia- bility in the controls. As with the model systems previ- ously mentioned, greater inhibition in the uncooked pork slices was achieved when the pH approached that of maximum NDMA formation, i.e. pH 3.5. At pH 5.6, a minimum ratio of 1:2 ascorbate/nitrite was necessary to obtain 80% 23 inhibition. In pork middles cured with brines containing the added amine and various ascorbate/nitrite ratios, ascorbate had its customary effect on the lean, but did not influence the N-nitrosamine content in the uncooked fat. The pork middles were then either canned and subjected to a 110°C heat process for 130 minutes, or were fried for 10 minutes after reaching 100°C, obtaining a final temperature of 140°C. The concentrations of NDMA in the fried lean did not differ appreciably from those in the canned lean. However, the concentration of NDMA in the fried fatty tissue was at least 10 times greater than the concentration in the lean with some of the ascorbate/nitrite ratios, thus confirming the model system work of Mottram and Patterson (1977). The authors also pointed out that the high solu- bility of NDMA in fat would make it less susceptible to volatilization. Fiddler 31 31. (1973) reported significant amounts of NDMA in 3 of 40 commercial frankfurters in concentrations ranging from 11 to 85 ug/kg. To study the effect of sodium ascorbate and its isomer sodium erythorbate, the nitrite level was increased by a factor of 10 in order to obtain a NDMA concentration of 10 ug/kg for the control. Sodium ascorbate or erythorbate completely inhibited the formation of NDMA in frankfurters for a normal 2 hour processing. Sen 31 31. (1973) attempted to inhibit N-nitrosamine forma- tion during the storage of curing spice mixtures containing 24 nitrite. The researchers reported ascorbate was ineffective and recommended the separate packaging of nitrite and spices. Ascorbic Acid Derivatives Bharucha 31H31. (1979) proposed that a good blocking agent in bacon should (i) serve as a NO radical trap; (ii) be fat soluble; (iii) be non steam volatile, and (iv) be stable up to maximum frying temperatures of about 174°C. These recommendations were based on the observa- tion that N-nitrosamine formation during the frying of bacon occurs almost entirely in the fat phase, and by a radical rather than an ionic mechanism. Ascorbic acid does not satisfy the second requirement, and as a result, may enhance N-nitrosamine formation in a predominently lipid two phase system (Mottram and Patterson, 1977), and is an undependable blocking agent in bacon (Sen 31 31., 1976). Consequently, Mottram and Patterson (1977) investigated the effect of ascorbyl palmitate on the N-nitrosation of PYR and dipropylamine in a two phase model system resem- bling adipose tissue. With corn oil as the nonpolar phase, ascorbyl palmitate inhibited the formation.of NPYR at the 90% level, while N-nitrosodipropylamine formation was inhibited by 20%. The authors theorized that the solu- bility of ascorbyl palmitate in the nonpolar phase allows it to be removed from contact with nitrite, and thus a 25 lower concentration of nitrogen oxides are produced in the aqueous phase. In addition, ascorbyl palmitate will be present to compete with the amines for any N-nitrosating species migrating to the nonpolar phase. Pensabene 31 31. (1976) employed a model system resembling bacon to examine the effect of NPYR formation of several lipophilic ascorbyl esters in combination with sodium ascorbate. The ascorbyl esters of oleic, palmitic, and lauric acids increased the inhibition of NPYR formation due to sodium ascorbate alone by about 15% in the aqueous phase. In the lipid phase, however, ascorbyl palmitate reduced the N-nitrosation of PYR by 48.6%, while the other two esters inhibited the reaction about 40%. Because these ascorbyl esters were only slightly fat soluble, the authors suggested that a larger reduction of NPYR formation could be achieved with a more lipophilic ascorbyl ester. Sen 31 31, (1976) treated bacon with ascorbyl palmi- tate, applying it as a spray just before frying. Ascorbyl palmitate was found to consistently inhibit the formation of NPYR in bacon above the 50% level, while sodium ascor- bate performed erratically and was less effective under similar conditions. Bharucha 31 31. (1980) achieved 70% inhibition of N-nitrosamine formation in fried bacon by the use of ascorbyl palmitate applied as a slurry in soybean oil just prior to frying. However, after ascorbyl palmitate was stored for three weeks, the inhibition fell to 50% when 26 applied at the same 500 mg/kg level. Long chain acetals of ascorbic acid were then examined by these authors, and found to be very effective at the 1000 mg/kg level. It was reported that the C12 and to some extent the C14 ascorbyl acetal left a soapy aftertaste that would make then unaccep- table in bacon. The °l6 ascorbyl acetal, however, left no such aftertaste and gave 80-90% inhibition of N-nitrosamine formation in the cook-out fat of bacon. This acetal was reported to be just as effective when sprinkled on bacon as a solid, and will retain its high blocking ability for at least 35 days under refrigerated conditions. It should be pointed out, however, that the acetals of ascorbic acid are not approved for food use at this time. Alpha-Tocopherol Alpha-tocopherol is another compound which fulfills the requirements for a good blocking agent as stated by Bharucha 31 31. (1979). In a mechanism analogous to the nitrite-ascorbic acid reaction, a-tocopherol reduces the N-nitrosating agent to a non-nitrosating species as outlined in Figure 2. Unlike many phenolic compounds, d-tocopherol cannot be C-nitrosated because of its fully substituted ring and thus cannot form a catalytic species (Walker t 1., 1979). Kamm t l. (1977) studied the reaction of a-toco- pherol with nitrite in an aqueous system, and reported that 27 cu: + 02+ .muwcuw: saw; Foswzaououie we covaommm O: .N mczmwd 010 O «:0 28 d-tocopherol reacted more rapidly with nitrite than did ascorbate at pH 2 or 3. At pH 5, however, a-tocopherol reacted with less than 5% of the nitrite present in an hour. Pensabene 31 31. (1978) experimented with a-toco- pherol in an aqueous model system and a two phase model system resembling bacon. a-Tocopherol was dissolved in the emulsifier Polysorbate 20 to facilitate its solubility in water and make it practical for use in a bacon cure. When the o-tocopherol/Polysorbate 20 ratio was 1:6, and a-tocopherol was used in the two phase model system at the 500 mg/kg levels, NPYR formation was reduced about 67% in the aqueous phase and 80% in the oil phase. Greater inhibition was achieved when the Polysorbate 20 concen- tration was reduced, but these emulsions were much less stable. Gray and Dugan (1975) achieved greater than 90% inhibition of the N-nitrosation of DMA using a-tocopherol in an aqueous model system. .Fiddler 31_31. (1978) investigated the effect of a—tocopherol on the formation of N-nitrosamines in fried bacon. The authors reported that the a-tocopherol/Poly- sorbate ratio had to be lowered to 0.4 in order to obtain optimum distribution of the blocking agent. a-toc0pherol by itself significantly inhibited the formation of NPYR in the bacon and cook-out fat. In addition, an a-toco- pherol-ascorbate combination treatment exhibited greater inhibition of the formation of NPYR and NDMA than did 29 ascorbate alone. In some cases, enhancement of NDMA formation occurred when the bacon was treated with ascor- bate. Walters §£.213 (1976) also examined the effect of a-toc0pherol on the production of N-nitrosamines in fried bacon. The authors reported a reduction of 82 and 62 percent of NPYR and NDMA respectively in the vapors when bacon lean was fried in fat containing 800 mg/kg of a-tocopherol. The blocking agent was also effective in lowering N-nitrosamine concentrations in bacon lean and cookout fat. A recent study by Reddy 31 31. (1982) investigated the feasibility of using a-tocopherol-coated salt as part of the dry cure for bacon. Approximately 96% inhibition of NPYR formation in the fried bacon was reported at the 500 mg/kg level of a-tocopherol, while in the cook-out fat, the NPYR concentration was reduced 92 percent. a-Tocopherol was not as effective in blocking the formation of NDMA, whose levels generally increase in the fried bacon treated with the a-tocopherol coated salt. Mergens 31 31. (1978) explored the subject of the stability of a-tocopherol in bacon. The average recovery of a-tocopherol immediately after processing was 85%. During storage, the average loss of o-tocopherol under refrigerated conditions was 3% per week, regardless of whether the packages were opened or closed. The overall recovery of a-tocopherol in the fried lean and cook-out 30 fat averaged just under 70 percent. Phenols Phenols are looked upon as unreliable blocking agents because of the abundance of seemingly conflicting data gathered on their inhibiting potential (Mirvish 31 31., 1975; Challis and Bartlett, 1975). The complexity of the situation is due to the existence of at least five dif- ferent reactions happening simultaneously (Douglass 31 31., 1978). The relative reactant concentrations and reaction conditions determine which mechanism will dominate, thus leading to a variety of results. Inhibition by phenols can occur either by the reduction of nitrite to a non N-nitrosating species (Figure 3, reaction 1, Challis and Bartlett, 1975), or by binding nitrite directly via C- nitrosation (Figure 3, reaction 2, Challis, 1973). How- ever, phenols have been observed to have a catalytic effect on amine N-nitrosation under some conditions. Walker 31 31. (1979) proposed the mechanism as shown in Figure 3 (reaction 3) to explain this phenomenon. The extent of catalysis will be dependent on the concentration of nitroso- phenol. Thus, the aerobic oxidation of nitrOSOphenol to the noncatalytic nitrophenol will influence the overall N-nitrosamine production (Davies and McWeeney, 1977). In addition, the uncatalyzed N-nitrosation of amines will also OCCUY‘. 31 .mp_cuw: new: mpocmca mo :o_wumwm .m mc:m_u afieoafioeoooiziz onzizufi + :ozflUfloI «:72: + onzioiz "one .ococnoaofcin 8. ...... Cu: + ONZIOIZ OAIIIOZO: + :Oznmvno Oz ANwas...coo-ooooooouoooooooo ON T- + “oz-:— 1... :0 :0 .3565: + ozu + I Nozzu + 32 Phenols were first mentioned as possible N-nitrosa- mine inhibitors by Bogovski 31.31. (1972). It was observed that when nitrite was added to apple juice in concentra- tions up to 100 mg/kg, some factor within the juice was tying up the nitrite, making it unavailable for detection. This factor was subsequently identified as tannin, a com- plex polyphenol found in high concentrations in apple juice. In a variety of acidic environments, tannins brought about greater than 95% inhibition of the N-nitro- sation of DEA and DMA. By varying the concentration of the reactants, the researchers were able to deduce that the phenols were competing with the amine for the N-nitro- sating species. Indeed, Challis (1973) determined that phenols react with nitrous acid about 104 times more rapidly than with DMA, and an even larger margin would exist for polyhydroxylated and polycyclic aromatic com-' pounds. However, Challis and Bartlett (1975) demonstrated that phenols such as 4-methylcatechol can act as powerful catalysts as well, especially when a high nitrite to phenol concentration ratio exists. Chlorogenic acid, structurally similar to this phenol and found in significant concentra- tions in coffee, exhibited a similar enhancement of N- nitrosation. Gray and Dugan (1975) examined the effect of tannic acid on the N-nitrosation of DNA in low moisture and aqueous model systems. At pH 3.5, greater than 95% 33 inhibition was achieved in both model systems, once the nitrite/tannic acid molar ratio was less than five. The same pattern of increasing inhibition of NDMA formation with increasing concentrations of tannic acid held true at pH 5.5. When propyl gallate was used in equimolar concen- trations as nitrite, almost complete inhibition of NPYR formation was achieved in a low moisture system under heat stress. The phenol was equally effective in a corn oil buffer system, as well as in an oil water mixture emulsi- fied with soluble starch. The antioxidants vanillin and thymol were only moderately effective in blocking NDMA formation. Hydroquinone, in equimolar concentration with nitrite, inhibited the same reaction at the 98% level in an aqueous system. Mirvish (1975) investigated the effect of tannic acid and its phenolic component, gallic acid, on the N- nitrosation of morpholine and piperazine. Gallate inhibited the N-nitrosation of morpholine by more than 95% over the pH range of l to 4, but was less effective when the concentration of the blocking agent was reduced four fold. Gallic acid was less effective in blocking the rapidly N-nitrosated piperazine, and became more ineffec- tual as the pH increased from 1 to 4. Tannic acid was not as effective in blocking the N-nitrosation of either amine. Pignatelli 31n31. (1976) also looked at the influ- ence of gallic acid on the N-nitrosation of DMA, with 34 radically different results. Gallic acid was shown to accelerate the formation of NDMA over a relatively narrow pH range around 4.0. A decreasing concentration of gallic acid resulted in increasing acceleration in an almost linear fashion. Coleman (1978) examined the influence of several phenolic compounds on NPYR proddction using a simple model system composed of methanol, nitrite, proline, and the blocking agent. Chlorogenic acid and caffeic acid increased the yield of NPYR substantially, while gallic acid was only moderately effective at the 60% inhibition level, but did remove all residual nitrite. The antioxi- dants butylated hydroxyanisole (BHA) and butylated hydroxy- toluene (BHT) were much less effective than gallic acid in limiting the yield of NPYR. Ethoxyquin, however, was effective in blocking the formation of NPYR while having little impact on residual nitrite levels, suggesting a different type of inhibitory mechanism from gallic acid. Davies and McWeeny (1977) examined the effects of various nitrosophenols on the rate of formation of NPYR at pH 5.0. It was demonstrated that the rate of N-nitrosation of pyrrolidine increased linearly with increasing concen- trations of p-nitroso-O-cresol. Two other nitrosophenols, p-nitrosothymol and l-nitroso-Z-naphthol also induced catalysis. Because no reaction takes place between p- nitroso-O-cresol and PYR in the absence of nitrite, it 35 was concluded that the mechanism does not involve a simple trans nitrosation from the nitrosophenol to PYR. At pH 5.0, a 2 to 1 ratio of nitrite/p-cresol in the presence of pyrrolidine resulted in 150% acceleration in the formation of NPYR. These researchers concluded that the overall effect of a phenol on amine N-nitrosation is dependent on which of several competing reactions dominate, dictated by reactant concentrations and pH; In a subsequent study, Davies 31 31. (1980) reported that the maximum formation of NPYR in the presence of nitrosocresol occurred at pH 5.0. Eleven compounds similar to nitrosocresol in structure were evaluated for catalytic potential. Only those nitrosophenols that could form quinonemonoximes or quinonemonoxime imines were able to accelerate the N-nitrosation of PYR. This conclusion was supported by the findings of Walker 31 31. (1979). m-Nitrosophenol, which cannot form a quinone tautomer, was shown to have no effect on the N-nitrosation of diethylamine. The researchers demonstrated the presence of p-nitrosophenol causes a radical modification in the reaction mechanism with a resulting shift from second to first order kinetics with respect to nitrite. 0n the basis of these discoveries, the mechanism given by reaction 3 in Figure 3 was proposed by the authors. Walker 31 31. (1982) demonstrated that 1,2 and 1,4 dihydroxyphenols (including naturally occurring flavonols) 36 inhibit N-nitrosamine formation at pH 4.0. However, 1,3- dihydroxyphenols are potent catalysts under similar con- ditions. This is attributed to the rapid formation of a nitroso intermediate which further reacts with a N-nitro- sating species to generate the powerful N-nitrosating agent. Knowles 31_31, (1975) positively identified twenty phenolic compounds in traditionally smoked bacon. An even greater number of phenolic compounds were present in bacon which was prepared by the application of liquid smoke condensates. Very little diffusion of phenols into the meat matrix was found in smoked bacon. However, nitrosa- tion of phenols from salivary nitrite to form the catalytic species remains a major concern. Sulfur Compounds Certain sulfur compounds have been reported to be effective N-nitrosamine inhibitors. Bisulfite reduces nitrite to nitrous oxide in a two step mechanism (reactions 18 and 19, Hisatune, 1961) while sulfamate reduces nitrite to molecular nitrogen (reaction 20, Jones, 1973) $0 + 2HNO -——-2NO + H 50 2 2 2 4 $02 + 2N0 + H20—"N20 + H2S04 (l9) NaNO + H Nso H—--NaHSO + N + H 0 (20) 2 2 3 4 2 2 37 Gray and Dugan (1975) examined the effect of several sulfur compounds on the N-nitrosation of secondary amines. These researchers obtained greater than 99% inhibition of the formation of NDMA with sodium bisulfite, once the bisulfite/nitrite ratio was greater than 2:1. While ammo- nium sulfamate blocked nitrosation of DMA at the 99% level at pH 3.5, its effectiveness decreased radically when a pH closer to that found in meat was used. The thiols, glutathione and cysteine were equally effective as ammonium sulfamate in blocking the formation of NDMA. In contrast to the action of ammonium sulfamate, the thiols effectiveness dropped only slightly as the pH was increased to 5.5. Sen and Donaldson (1974) reported on the influence of glutathione on the formation of mononitrosopiperazine and dinitrosopiperazine from a piperazine containing drug in a human gastric juice environment. Glutathione was found to be more effective than ascorbic acid in blocking mono- nitrosopiperazine formation, and did not catalyze the formation of dinitrosopiperazine as ascorbic acid had. Davies 31 31. (1978) investigated the competitive nitrosations of cysteine, p-cresol, and PYR in an aqueous solution. The S-nitrosation of cysteine was found to proceed faster than phenol C-nitrosation, which in turn was faster than the N-nitrosation of PYR over the pH range of 3.0 to 5.5. These data do not insure that cysteine will 38 inhibit N-nitrosamine formation, because thiols, like phenols, can form nitroso compounds which can themselves act as nitrosating agents. Indeed, cysteine causes an eleven fold increase in NPYR after 24 hours in an aqueous environ- ment (pH 5.0). However, at pH 3.0, cysteine effects greater than 94% inhibition of NPYR formation. The authors attributed this last result in part to the decomposition of excess nitrite at this lower pH. In a similar study, Massey 31 31. (1978) investigated the competitive nitrosa- tion of PYR, ascorbic acid, cysteine, and pecresol in an aqueous and protein based model system. Of the two control reactions, the protein based model system gave lower NPYR concentrations, accompanied by significantly lowered. cysteine, lysine, and tyrosine residues. Cysteine did reduce the rate of Nenitrosation of PYR in both model systems at pH 5.25. However, for the reactions involving cysteine, the rate of N-nitrosation of PYR was higher for the protein based model system, despite the fact that the residual nitrite levels were half as much as in the aqueous system. Antioxidant Activity of Soybeans The expensive safety testing of synthetic antioxidants required by the federal government has prompted many food manufacturers to look towards natural food sources for antioxidants (Marshall, 1974). Soybean products, 39 particularly soybean flour, have been used effectively as antioxidants in a variety of food products ranging from lard to frozen raw ground pork (Musher, 1935; Neill and Page, 1956). For example, Overman (1951) reported that pastry containing low fat soy flour at the 10% level extended the storage life of the pastry three fold before the development of organoleptic rancidity. The effective- ness of soy flour as an antioxidant is determined by the heat treatment received by the flour, the oil level in the flour, the character of the fat or oil in the food product, and the nature of other product ingredients (Hayes _3._1., 1977). Several constituents of soy flour have been reported to be effective antioxidants. One group of these consti- tuents is the isoflavone glucosides, genistin and daidzin, whose structure are shown in Figure 4 (Pratt and Birac, 1979). These compounds can undergo enzyme glycoside hydrolysis or acid glycoside hydrolysis to form the aglucone genistein and glucose and the aglucone daidzein and glucose. Other isoflavone glucosides have been identified (Gyorgy 31 31., 1964; Okano and Beppu, 1939), but genistin and diadzin make up approximately 85% of this class of phenols (Naim.31.31., 1974). There are also flavones, which are isomeric with the isoflavones. Both are included in the general class of water soluble phenolic glucoside pigments known as flavonoids (Harborne, 1965). Okano and Beppu 40 .mmcwmoozpm m=o>epeowF gowns mew mo mczuuzcum .e mesmwm : "we 5:8 .1... 62386-6...» 532...... z "a: .z u I. 53.3 :o "a: .: n E 5.3.36 0 a: O 0|.6 41 (1940) reported isolating three flavones from soybeans. Watts (1962) has identified the plant flavonoids as among the most potent of the phenolic antioxidants. How- ever, there are contrasting views as to what form of the flavonoids are the most effective antioxidants and what is their mode of action. Pratt and Watts (1964) reported that the glycoside and aglycone species of isoflavone possessed similar antioxidant activity. Gyorgy 31_31: (1964), on the other hand, found that the aglucones isolated from fermented products were superior in antioxidant activity to the extract obtained from control soybean whose isoflavones would presumably have the glycosidic linkage still intact. Pratt (1972) reported that flavonoids are primary antioxi- dants whose main role is that of a free radical acceptor rather than a metal scavenger. Gyorgy_31.311 (1964), how- ever, speculated that the flavonoids acted via protection and preservation of vitamin E rather than direct biological action. Another class of compounds in soy flour reported to possess antioxidant properties are the phospholipids. In a study carried out by Dahle and Nelson (1941), it was reported that the phospholipid fraction and an ethanol extract exhibited a stronger effect in dry, fresh milk than did either the aqueous, acetone, ether, or hexane extract of soyflour. Schwab 31_31: (1950) indicated that the anti- oxidant effect of phosphatides was due in part to the metal 42 scavenging ability of the phosphoric component, which would preserve the primary antioxidant present. Amino acids may be antioxidative under one set of con- ditions, but prooxidative or borderline under another set of conditions (Hayes 31 31., 1977). Marcuse (1960) demon- strated that 10 of the 11 amino acids studied had antioxi- dative effects of varying degree. The effect was strongest with histidine, while cysteine was the one prooxidative amino acid. The amino acids were able to act as primary antioxidants by themselves and were synergistic with o-tocopherol. With increasing concentrations of the amino acids, they become prooxidative, after going through a maximum antioxidative effect. Bishov and Henick (1975) evaluated the antioxidative activity of the amino acids cysteine, methionine, proline and phenylalanine. All four acted as primary antioxidants, and their activity was only slightly concentration dependent. Hydrolyzed soy protein, consisting of peptide and amino acid mixtures, also were effective antioxidants and were synergistic with phenolic antioxidants such as a-tocopherol. The authors reported that the synergistic effect was not due solely to the inactivation of prooxidative metals, and that the mechanism still remained to be established. EXPERIMENTAL Important safety note: Caution should be exercised in the handling of N-nitrosamines since they are potential carcino- gens. Direct contact with these chemicals should be avoided. Safety gloves should be worn whenever N-nitrosa- mines are being handled. All experimental work should be done in a hood or a well-ventilated area. Materials All chemicals and solvents employed were of analytical grade and used without further purification. Sodium nitrite, citric acid, sodium ascorbate, dibasic sodium phosphate and all of the solvents were purchased from Mallinckrodt Inc. (Paris, KY), except where specified. NPYR, PYR, rutin, quercetin dihydrate, resorcinol, and Biochanin A were purchased from Aldrich Chemical Co. (Milwaukee, WI). ICN Pharmaceuticals Inc.(Plainview, NY) provided the dehydroascorbic acid, ascorbic acid, ascorbyl palmitate, and genistein. €15 ascorbyl acetal was donated by Canada Packers Inc. (Toronto, Ontario, Canada), and ethoxyquin was obtained from Pfaltz and Bauer Inc. (Stam- ford, CN). Pure corn oil was purchased from General Food Stores at Michigan State University (East Lansing, MI). 43 44 Pork back fat, pork, and lean beef were obtained from local suppliers, while the defatted soy flour, labeled Soyafluff 200, was donated by Central Soya Co. (Fort Wayne, IN). Methods Aqueous Model Systems Model systems containing potential blocking 33ents The aqueous model system studies were performed in a citrate-phosphate buffer of pH 3.5 or pH 5.5, prepared according to the specifications of Gomori (1955). Using a 0.1M solution of citric acid and a 0.2M solution of dibasic sodium phosphate, 34.9 ml of citrate solution and 15.1 ml of phosphate solution were combined per 100 ml of buffer to obtain a pH of 3.5. To prepare a pH 5.5 buffer, 21.6 ml of citrate solution and 28.4 ml of phosphate solution were combined per 100 ml of buffer. The pH of the aqueous model systems was monitored using a digital pH meter (Model 601A, Orion Research Inc., Cambridge, MA). In order to examine the effect of blocking agents on the N-nitrosation of PYR, 2.0 mM of nitrite and 1 mM of PYR were used in this series of aqueous model systems. The reactants were made up in stock solutions where possible with citrate phosphate buffer. Ascorbic acid was the only potential blocking agent whose water solubility was great enough to be added in this manner. All other blocking 45 agents were weighed out on an analytical balance and trans- ferred directly to a stoppered 125 ml Erlenmeyer flask. The reactants which could be pipetted were transferred into a 50 ml volumetric flask and made to mark with buffer. Once the volume had been standardized, the mixture was then poured into the reaction flasks and the pH adjusted with concentrated HCl or NaOH. Various concentrations of the blocking agents were used, each one being prepared in dupli- cate. Once the samples were assembled, they were immediately placed in a Dubnoff Metabolic Shaking Incubator (GPA Pre- cision Scientific Co., Chicago, IL) set at 80°C for two hours. Upon completion of the reaction time, the samples were plunged in an ice bath to terminate further reaction. The reaction mixture was then transferred to a separatory funnel and extracted with two 25 m1 aliquots of methylene chloride. The extract was collected in a Kuderna Danish evaporator apparatus (Kontes Glass Co., Vineland, NJ), and the volume was reduced to l or 2 m1. A small quantity of methylene chloride was used to rinse the apparatus, and the solution was transferred to a 5 m1 volumetric flask and made to mark. Due to the prohibitive cost of pure genistein and Biochanin A, all concentrations and reactant volumes were reduced by a factor of five for these aqueous model systems. The glassware size was also reduced accordingly. 46 Model systems containing defatted soy flour Samples were prepared using 130 ml of pH 3.5 citrate-phosphate buffer (see previous section). PYR and nitrite were made up in stock solutions identical in con- centration to the stock solutions formulated in the pre- vious section. The concentration of nitrite was varied while each sample contained 1 mM of PYR and 20 g of defatted soy flour. The reaction flasks were immersed in the shaking water bath at 80°C for 2 hours. Each level of nitrite was run with a control containing no soy flour. Upon completion of the reaction, 10 ml of 1N NaOH were poured into the reaction flasks, and the samples were then transferred into 500 ml round bottom flasks containing 50 g of NaCl and boiling chips. The reaction flasks were rinsed with 200 ml of distilled water, which was combined with the samples, and steam distillation was performed. The distillate (250 ml) was then collected and extracted with two 50 m1 aliquots of methylene chloride. The extract was tranSferred to a 100 ml volumetric flask and made to mark. Due to the sensitivity of the TEA system, the samples were ready for injection at this point without further concen- tration. Model systems containing methanol extract and residue of defatted soy flour A methanol extraction of defatted soy flour was executed according to the procedure of Walter (1941) in an 47 effort to isolate the glucoside isoflavones of soy flour. Twenty grams of defatted soy flour were refluxed for 90 minutes in 200 ml of methanol. This solution was divided equally between two 500 m1 centrifuge bottles and spun at 5000 rpm for ten minutes using a Model K, IEC International Centrifuge (International Equipment Co., Boston, MA). The methanol extract was then decanted into two more 500 ml centrifuge bottles and the residue was saved. Approximately 100 ml of acetone was added to each bottle, precipitating some of the phosphatides, which carried with them carbo- hydrates, saponin, and other impurities (Walter, 1941). The two extracts were again centrifuged at 5000 rpm for 10 minutes and decanted into a round bottom flask, while the residue was combined with the residue from the first centrifugation. The methanol-acetone mixture was evapo- rated at 40°C in a rotary evaporator (Buchi Instrument Co., Switzerland), leaving a yellow oil. The oil and the combined residual soy flour were left overnight at room temperature to remove any remaining solvent. The soy flour residue and the concentrated methanol extract were each dissolved in 100 ml of pH 3.5 citrate-phosphate buffer and transferred to a 250 m1 stoppered Erlenmeyer flask. PYR and nitrite were made up in stock solutions as previously described. The concentration of nitrite was varied, while each sample received 1 mM of PYR, and the total volume of buffer was brought up to 130 m1. For each extract and 48 residue pair, a control was run, and all samples were prepared in duplicate. The heating of the samples, steam distillation, and extraction procedure was identical to that described in the previous section. Lipid Model §ystems Ground adiEose tissue system In these model systems, large glass ampules were employed as reaction flasks, and ground pork back fat served as the reaction medium. The adipose tissue was first ground in a food grinder (Oster Corporation, Milwau- kee, WI). Using an analytical balance, 1 mM of sodium nitrite was carefully weighed into the glass ampules, followed by 8 g of ground adipose tissue. This was done by rolling the ground fat into long narrow cylinders with wax paper, freezing them on dry ice, and slipping them into the small opening of the ampule. The required amount of blocking agent to be tested was poured as quantitatively as possible into the ampule using a glass funnel. Just prior to sealing the glass ampule, 0.5 mM of PYR was injected into the sample using a 50 ul syringe. Each concentration of blocking agent was investi- gated in duplicate. The ampules were subsequently heated . in a shaking bath at 80°C for two hours. Upon completion of the reaction time, the ampules were frozen in dry ice to stop further reaction. The ampules were then crushed 49 with a large mortar and pestle containing 100 ml of distilled water. After the glass and adipose tissue had been thoroughly ground, the sample was poured into a Buchner funnel containing #1 Whatman filter paper. The mortar and pestle were rinsed with 200 m1 of distilled water, which was then poured over the residue in the funnel. The filtrate was transferred to a 500 ml separa- tory funnel, and extracted with a 100 ml aliquot of methy- lene chloride, followed by a 50 ml aliquot. The extract was poured directly into a Kuderna Danish evaporator, and concentrated to 5 ml. This solution was then analyzed by gas chromatography. Corn oil-buffer model gystem A predominantly lipid model system resembling bacon fat was employed essentially using the procedure described by Mottram and Patterson (1977). In a stoppered Erlenmeyer flask, 100 ml of pure corn oil were mixed with 15 m1 of a pH 5.5 sodium citrate-citric acid buffer. The buffer was formulated by adding 14.8 ml of a 0.114citric acid solution to 35.1 ml of a 0.lllsodium citrate solution and diluting to 100 ml with water. Sodium nitrite and PYR were again made up in stock solutions of buffer to facilitate the pipetting of 0.25 mM of sodium nitrite and 0.125 mM of PYR into the reaction flasks. The blocking agents (0.50 mM) were weighed out and transferred directly to the reaction flasks. The reactions were carried out at both 25°C and 50 80°C, with each sample being prepared in duplicate. Upon completion of the two hour reaction time, the reaction was quenched using 5 ml of 1N NaOH solution. The reaction mixture was then poured into a 500 m1 round bottom flask containing 50 g of NaCl and boiling chips. The reaction flasks were rinsed with 120 ml of distilled water, which was added to the round bottom flask. Steam distil- lation was then performed and 130 ml of distillate were collected. The distillate was then extracted with two 25 ml aliquots of methylene chloride. This solution was then injected into the TEA system for analysis. Because of the prohibitive cost of pure genistein, only one test was conducted at each temperature along with a control. All reactant concentrations and solution volumes were reduced by a factor of five for this model system. The size of the glassware was modified accordingly. Frankfurter Study Frankfurters were prepared by combining 10.2 pounds of a lean beef mixture and 15.5 pounds of a fatty pork blend for 30 pounds of batter. Representative samples from each block were analyzed for percent moisture and fat using standard analytical procedures (AOAC, 1975). Formu- lations based on the analysis of the raw materials were computed to yield frankfurters with 26% fat and 10% water in the finished product. Appropriate amounts of the meat 51 blocks, spices, and ice were combined in a vertical cutter mixer (Model VCM 40E, Hobart Manufacturing Co., Troy, OH). In addition,156 mg/kg of sodium nitrite and 1000 mg/kg of dimethylamine hydrochloride were added, and the mixture was blended for five minutes. The batter was then divided into four aliquots, and each batch was transferred to a table cutter (Model 841810, Hobart Manufacturing Co., Troy, OH) and blended for 30 seconds with either 0%, 2%, 4%, or 6% soy flour. Water was added to each of the three batches blended with soy flour to maintain a consistent percentage of moisture between the treatment levels. The batter was transferred to a water pressure sausage stuffer, stuffed into 24 mm frankfurter casings, linked, and cooked in a smokehouse equipped with temperature and humidity controls (Drying Systems, Inc., Chicago, IL). No smoke was added during the heat cycle process of 10 minutes at 62.8°C dry bulb (DB), 23% relative humidity (RH); 15 minutes at 71°C DB, 28% RH; 35 minutes at 88°C DB, 35% RH, followed by a cold water shower of 7 minutes. The nitrite concentrations of the frankfurters were analyzed using the standard AOAC procedure (AOAC, 1975). The frankfurters were either left unheated or were cookedfbr 5 minutes in boiling water. The concentration of DMNA in the frankfurters was determined using essen- tially the TEA procedure of Gray et a1. (1981). 52 Analytical Techniques GC analysis of N-nitrosamines The quantitative and qualitative determination of NPYR was performed on a Hewlett-Packard Model 5830A Gas Chromatograph (Corvallis, OR) equipped with a flame ioni- zation detector (F10) and a Hewlett-Packard Model 18850 Terminal which integrated FID response to peak area. Linearity of response was established using standard solu- tions of varying concentrations. Standard curves were prepared for calculating the concentrations of NPYR in the samples. At least three injections were made for each sample. The oven temperature of the GC was 180°C, while the injection temperature and F10 temperature were 200°C and 250°C, respectively. The column packing material was a 10% Carbowax 20M-5% KOH on 80/100 Chromosorb WAW packed into a 2mx4mm glass column (Supelco Co. Inc., Bellefonte, PA). TEA analysis of N-nitrosamines The qualitative and quantitative analyses of NPYR were also performed on a Model 3700 Varian Gas Chromato- graph (Varian Co., Walnut Creek, CA) equipped with a Model 502 TEA detector (Thermo Electron Corporation, Waltham, MA). A Hewlett-Packard 3390A Integrator completed the analytical system. The glass column (2mx2mm) was packed with 10% Carbowax 20M and 5% KOH on Chromosorb WAP 80/100 mesh (Supelco Inc., Bellefonte, PA). The oven temperature and 53 injection temperature of the GC was 180°C and 200°C respec- tively. 0n the TEA, the oxygen flow was 10 ml/min while the nitrogen carrier gas flow was 35 ml/min. The pyrolyzer temperature was 450°C. Again, at least three injections were performed for each sample and a standard curve was prepared by injecting three standards of known concentra- tions. HPLC analysis of soybean isoflavones The qualitative analysis of the methanol extract of soy flour for the aglucone isoflavones was performed on a Model ALC-201 High Pressure Liquid Chromatograph equipped with a Model 440 Absorbance Detector (Waters Associates, Milford, MA) and a U6K loop injector. The sample was prepared by dissolving the yellow oil left after concentra- ting the methanol extract from 20 grams of defatted soy flour in 10 m1 of methanol and 5 ml of 6% HCl solution. This mixture was heated on a steam bath for 45 minutes in order to hydrolyze the bond between glucose and the iso- flavone. The resulting solution was extracted with two 5 ml aliquots of chloroform, and the combined chloroform extract was back extracted with two 5 ml aliquots of methanol to remove more of the carbohydrate impurities. The column was a 50 cm x 3 mm Partisil PXS 10/50 (Whatman Inc., Clifton, NJ). The mobile phase was chloroform pumped at a flow rate of 1 ml/min. The UV detector was set at 254 nm, R = .05. 54 The sample was injected in 50 ul aliquots at least three times. A pure sample of genistein was dissolved in chloroform and injected into the HPLC to determine the identity of peaks from the methanol extract. RESULTS AND DISCUSSION Aqueous Model §ystems Model Systems ContainingyPotential Blocking Agents A series of model system experiments was performed to investigate the effect of several N-nitrosamine blocking agents in an aqueous environment of pH 3.5 and 5.5. The lower pH was used because it is the pH at which maximum N-nitrosamine formation takes place (Mirvish, 1975), while the latter is generally recognized as the pH of a meat system. Because there has been many previous studies on the effect of ascorbic acid on N-nitrosamine formation (Mirvish 31 31., 1972; Kawabata _1 _1., 1974; Gray and Dugan, 1975), this blocking agent was used to confirm the efficacy of the model system. Quercetin dihydrate and rutin are two flavones whose phenolic constituents make them structurally similar to the principal isoflavone of soy flour, genistein. The flavonoid compounds in soy flour and structurally similar antioxidants are of primary interest in this study because of the possible use of soy flour as a means of blocking N-nitrosamine formation in food products such as frankfurters. The employment of blocking agents of natural origin such as soy flour is 55 56 advantageous because the expensive safety testing required of a new synthetic blocking agent is avoided. Ethoxyquin is an antioxidant which has been reported to be effective in blocking N-nitrosamine formation in a methanol model system (Coleman, 1978). Ascorbic acid was investigated over a wide range of concentrations in order to examine its effect on the N- nitrosation of PYR. At pH 3.5, an equimolar concentration of ascorbic acid and nitrite was sufficient to obtain greater than 95% inhibition of NPYR formation (Table 1). Gray andDugan (1975) also obtained greater than 95% inhi- bition of the formation of NDMA using equimolar concentra- tions of nitrite and ascorbate in an aqueous model system at pH 3.5. The pKa of DMA and PYR and the rate constants (k2) of their N-nitrosation reactions are relatively close (Mirvish 31.31.. 1972), and therefore one would expect the inhibitory effect exerted by ascorbic acid on the N-nitro- sation of these amines to be similar. Mirvish 31 31. (1972) investigated the effect of ascorbic acid on the N-nitrosation of a variety of amines at several values of pH. These researchers obtained greater than 95% inhibition of NDMA formation employing an ascorbate/nitrite concen- tration ratio of 2:1 in the presence of an excess of amine at pH 4.0. Kawabata 31 31. (1974) obtained enhancement of NDMA formation when the nitrite concentration was ten times 57 Table 1. Effects of ascorbic and dehydroascorbic acid on N-nitrosamine formation in an aqueous model system (pH 3.5) containing PYR (1 mM) and NaNOz (2 mM). Blocking Concentration % Inhibition agent (mM) PYR .5 NPYR Ascorbic acid 0.10 24.0 0.25 33.0 0.50 49.8 1.00 74.8 2.00 ' 98.7 10.00 100.0 Dehydroascorbic acid 1.00 -1o.9a aThe negative sign indicates an enhancement of the N-nitro- sation reaction. 58 greater than ascorbate in an aqueous model system at pH 6.0. In Table l, a 20:1 ratio of nitrite to ascorbate resulted in approximately 25% inhibition of the reaction, and no enhancement of NPYR formation occurred at any con- centration of ascorbic acid at pH 3.5. The apparent discre- pancy is undoubtedly due to the differences in pH of the aqueous systems. Kawabata 31_31, (1974) obtained no enhancement of NDMA formation at pH 3.6 for any concentra- tion of ascorbate. Mirvish _1 _1. (1972) determined that ascorbic acid can have an acceleratory effect on NDMA formation, but because they were interested in i vivo N-nitrosation, extremely low values of pH were employed (pH 1 and 2). Dehydroascorbic acid demonstrated a small catalytic effect on the N-nitrosation of PYR at a concentration ratio of nitrite/dehydroascorbic acid of 2:1. This is in agreement with the reaction mechanism depicted in Figure l, in which dehydroascorbic acid is the oxidized product of the conversion of nitrous anhydride to nitric oxide. It is evident that only the reduced form of ascorbic acid can be an effective inhibitor. The catalytic effect of dehydro- ascorbic acid on the N-nitrosation of PYR may be the result of the formation of radical species described by Yano 31 31. (1976). These researchers obtained stable radical products upon heating a mixture of dehydroascorbic acid with amino acids in water or ethanol. A possible mechanism of 59 catalysis is the formation of a transnitrosating species from the stable radicals. The influence of quercetin dihydrate on the N-nitrosa- tion of PYR is shown in Table 2. This compound demonstrated‘ only limited solubility in the aqueous model system, forming a coarse dispersion. At low concentrations of the anti- oxidant, quercetin has a substantial accelerative effect on the N-nitrosation reaction when used in aqueous systems. Interestingly, this occurs at both values of pH, and may be attributed in part to the formation of the catalytic nitroso intermediate proposed by Walker 31 31. (1982). This theory is described by the mechanism depicted in Figure 5, in which a nitroso intermediate is formed at high ratios of nitrite/phenol concentrations. This intermediate is a more powerful N-nitrosating agent than p-nitrosophenol (Walker _1 _1., 1982). From the mechanism, it is apparent that only the 1,3 dihydroxyphenol will form the catalytic species, while the 1,2 and 1,4 dihydroxyphenols will not enhance the N-nitrosation reaction. An examination of the structure of quercetin dihydrate reveals that this compound possesses both a 1,3 and a 1,2 dihydroxyphenolic ring (Figure 6). At each pH, the catalytic effect of the phenol is quickly eliminated as its concentration is increased. The inhibition is probably the result of the formation of C-nitrosophenols, which would tie up some of the free dinitrogen trioxide. Also, the reduction of dinitrogen 60 Table 2. Effect of quercetin dihydrate on N-nitrosamine formation in an aqueous model system containing PYR (1 mM) and NaN02 (2 mM). Concentration of ‘ % Inhibition quercetin dihydrate PYR + NPYR (”“1 pH 3.5 pH 5.5 0.1 -93.3a -138.7a 0.5 41.4 71.3 1.0 65.0 72.0 2.0 73.1 71.4 5.0 89.6 68.2 aThe negative sign indicates an enhancement of the N-nitrosation reaction. 61 - . .Ammmp ..Im 1m to c xxocvxgwu m P >a comucEcow mcpsmmocgwciz co mwmxpmumu ho “a_cmuwmnvuwwwmwum 3630.535 Dachau: toga-sumac :62 :oz 6 . w o . + «oz: IMV + ozzafi :z .m ouz.o.z :02 o :0 . .0 :02 I «Cu: + :02 O .m «gnaw; 62 Quercetin dihydrate Rutin 01-1 0' ‘ 01-1 0 (C I °°12H21°9 on HO OH Ethoxyquin Genistein '1‘ N ci-i3 cwgcwzo " CH3 C1 6 L-Ascorbyl acetal cuaicw2114cw HN02 + L—Q-COOH | 0 Fl Q—COOH + NO-——-> (“j-00011 1 . 01:: C) I I Decarboxylation hi 1 01:: C) Figure 7. Postulated free radical mechanism of N-PYR formation (Bharucha 31 31., 1980). 87 in the two phase model system to compare its influence on NPYR formation with that of the flavonoid compounds. The meta dihydroxyphenol, like the flavonoid compounds, had little effect on NPYR formation at 25°C, but did induce approximately the same moderate acceleration of the N-ni- trosation reaction at 80°C. The enhancement of formation by flavonoids in the two phase model system is most likely analogous to the catalytic mechanism of sodium ascorbate proposed by Mottram and Patterson (1977). The flavonoid compounds are more soluble in the aqueous phase and are available to reduce dinitrogen trioxide to nitric oxide. Nitric oxide and other nitrogen oxides generated from nitric oxide are free to migrate into the nonpolar phase, where they can rapidly react with secondary amines. The mono and dihydroxy rings of the flavonoid compounds are susceptible to C-nitrosation. The mechanism of this C- nitrosation, however, is unknown. The binding of the N- nitrosating species to the flavonoid compound may explain the much smaller catalytic effect exerted by the flavonoid compounds in comparison to the acceleration induced by sodium ascorbate. Frankfurter Study The frankfurter study was performed to investigate the effect of soy flour on NDMA formation in this cured meat product. During processing of the frankfurters, the batter 88 was spiked with 1000 mg/kg of DMA. The frankfurters from each treatment level were analyzed for residual nitrite after processing (Table 13). The control frankfurters contained the lowest residual nitrite, which is in agreement with the data of Sofos 31 31. (1977). These authors observed increasing residual nitrite levels with increasing soy levels in the formulation of the frankfurters, explain- ing that soy proteins probably do not bind as much nitrite as meat proteins. However, in the present study, the residual nitrite decreased in almost a perfectly linear fashion as the levels of soy in the formulation increased. The results of the NDMA analyses of the unheated and heated (boiled) frankfurters are presented in Table 14. The soy flour was not effective in inhibiting the formation of NDMA in the frankfurters. In general, the levels of NDMA increased with increasing levels of soy flour. The enhancement of NDMA formation was more pronounced in the frankfurters which had been boiled for 5 minutes. A possible explanation of the enhancement of NDMA formation involves the aglucone isoflavone genistein. Considering the 4% soy flour treatment level for example, the nitrite/ genistein concentration ratio would be approximately l400:1, assuming no genistin is hydrolyzed to genistein and glucose during the heat treatments. This assumption is not a valid one, and consequently the concentration ratio would be somewhat lower. Nevertheless, a high concentration of 89 Table 13. Nitrite contents of frankfurters prepared with varying levels of soy flour. Frankfurter NaNOZ (mg/kg) sample Control 66.3 2% soy flour 92.4 4% soy flour 86.6 6% soy flour 78.0 90 Table 14. Effect of increasing levels of soy flour on the N-nitrosamine content of frankfurtersa. Sample Concentration of DMNA % Ingibitionb (mg/kg) DMA + NDMA l) Unheated franks control 1.07 - 2% soy flour 1.01 5.6 4% soy flour 1.11 -3.4 6% soy flour 1.45 -35.5 2) Heated franks control 1.36 - 2% soy floor 1.82 -33.4 4% soy flour 2.75 -101.8 6% soy flour 1.98 -45.2 aUnheated frankfurters refers to frankfurters that are cooked during processing. Heated frankfurters are those that are heated in boiling water for 5 minutes. bThe negative sign indicates enhancement of the N-nitrosa- tion reaction. CThe original emulsion was spiked with 1000 mg/kg DMA and 156 mg/kg NaNO2 was added. nitrite in comparison to the genistein may well be respon- sible for the catalytic effect of soy flour via the mecha- nism proposed by Walker _1 31. (1982). In a study by the Nitrite Safety Council (1980), beef, pork, and cheese frankfurters were cooked by several methods and analyzed for NPYR, NDMA, and N-nitrosomorpho- line. With few exceptions, non-detectable levels of N- nitrosamines were reported. Only fried frankfurters yielded trace amounts of NDMA, which was defined as l to 5 ug/kg. Holland 31 31. (1981) investigated N-nitrosamine levels in frankfurters purchased at the retail level in Canada. 0f the 24 samples tested, only 25% were found to contain over 5.0 ug/kg DMNA, and the highest concentration of this N-nitrosamine was 14 ug/kg. Similar levels of N-nitrosomorpholine were also reported by these investiga- tors, while only trace Concentrations of N-nitrosodiethyla- mine, N-nitrosobutylamine, NPYR, and N-nitrosopiperidine were detected. The results of the study by Holland _1 _1. (1982) indi- cate that N-nitrosamine levels in frankfurters are approxi-~ mately ten thousand times lower than those found to induce cancer in laboratory animals. The risk of botulism. how- ever, would be a real danger should nitrite be eliminated from these cured products. SUMMARY AND CONCLUSIONS The effectiveness of a number of potential N-nitrosa- mine blocking agents was investigated in aqueous and lipid model systems. Soy flour was examined to ascertain its potential as a blocking agent in an aqueous model system, and was employed in a frankfurter batter spiked with amine to evaluate its effectiveness as an N-nitrosamine blocking agent in this cured meat product. Ascorbic acid was effective in inhibiting NPYR forma- tion in an aqueous model system, but greatly enhanced the N-nitrosation reaction in a two phase lipid-containing model system. In this system, the lipophilic derivatives of ascorbic acid, ascorby1 palmitate and ascorbyl acetal, were effective blocking agents at ambient temperature. The flavones quercetin dihydrate and rutin were moder- ately effective in blocking NPYR formation in the aqueous model systems. However, enhancement of the N-nitrosation reaction resulted when the concentration ratio of nitrite/ flavone was high. In the two phase model system, the flavones exerted little influence on the N-nitrosation reaction at 25°C, while substantial acceleration of NPYR formation was induced at 80°C. The isoflavone, genistein, demonstrated a mildly catalytic effect in both model 92 93 systems. The antioxidant ethoxyquin almost totally inhibited the formation of NPYR when used at high concentrations in the aqueous model system, but accelerated N-nitrosamine forma- tion when used at low concentrations. In the two phase model system, ethoxyquin exerted a mild catalytic effect on NPYR formation at ambient temperature, but was the only potential blocking agent to partially inhibit the N-nitro- sation reaction at 80°C. Soy flour was highly effective in blocking NPYR forma- tion at a variety of nitrite levels in an aqueous model system. The methanol extract containing the phenolic constituents of soy flour was not as effective in blocking the N-nitrosation of PYR as the residue remaining after methanol extraction of the soy flour. Soy flour slightly enhanced the formation of NDMA when included in a frank- furter emulsion spiked with amine, and this enhancement became more pronounced after the frankfurters were heated for 5 minutes in boiling water. As a result of these studies, several conclusions can be drawn. These are summarized below: 1. Ascorbic acid is an effective blocking agent of NPYR formation in an aqueous model system, but is catalytic in an aqueous-lipid model system. 2. Lipophilic ascorbic acid derivatives effectively block NPYR formation in a two phase model system for two 94 hours at 25°C, but not for two hours at 80°C. 3. The flavonoid compounds, quercetin dihydrate, rutin, and genistein enhance NPYR formation when present in an aqueous model system at low concentrations, but exert a greater inhibitory effect as their concentration increases. 4. Ethoxyquin is a more effective blocking agent at 80°C than at 25°C in a two phase model system. 5. Soy flour is an effective blocking agent of NPYR formation in an aqueous model system. This influence is partially due to the phenolic compounds of soy flour, but the remaining constituents play a greater inhibitory role. 6. Soy flour is not a dependable N-nitrosamine blocking agent in lipid systems, and therefore its use as such in frankfurters is not recommended. PROPOSALS FOR FURTHER RESEARCH The examination of the blocking potential of a variety of compounds in model and meat systems has raised questions which merit further study. These include: 1. The stability of the ester and ether linkages of ascorbyl palmitate and ascorbyl acetal respectively under prolonged heat stress. 2. The effect of higher levels of flavonoids on the N-nitrosation of PYR in the two phase model system. 3. The effect of ethoxyquin on the N-nitrosation of PYR in the two phase model system at 120°C for 2 hours, allowing the aqueous phase to volatilize off. 4. The effect of the methanol extract of soy flour on the N-nitrosation of PYR in the two phase model system. 5. The effect of soy protein isolate on the N-nitro- sation of DMA in frankfurters. 6. 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