h- F PLACE IN RETURN BOX to remov Michigan State RM w“I LIBRARY University fl 0 this checkout from your record. TO AVOID, F INES return on or before date due. DATE DUE DATE DUE DATE DUE l___ f“ [\ MSU Is An Affirmative Action/Equal Opportunity Institution emu-neon»! The Influence of Microwave Beating on the Formation of N-Nitrosamines in Bacon by SHAUN CHENGHSIUNG CHEN A THESIS submitted to MICHIGAN STATE UNIVERSITY in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE SCHOOL OF PACKAGING 1991 ABSTRACT The Influence of Microwave Beating on the Formation of N—Nitrosamines in Bacon by SHAUN CHENGHSIUNG CHEN Bacon was prepared using conventional frying and microwave cooking in transparent and susceptor packages in a 700 W microwave oven. Bacon slices were prepared to three degrees of doneness, undercooked, overcooked and similar to standard pan frying (3 min on each side at 340°F), by controlling the time in the microwave oven. The package interfacial temperature and bacon temperature were monitored using a Lnxtron fluoroptic probe system. Final bacon temperatures of 167°C (330°F) and 143°C (290°F) were achieved for bacon samples cooked to the same degree of doneness as fried.bacon.in the susceptor (2.5 min) and.transparent (3 min) packages, respectively. Compared to frying, less N- nitrosopyrrolidine (NPYR) was present in bacon prepared either in the transparent or susceptor packages. Greater concentrations of NPYR were found in bacon prepared on the susceptor trays compared to bacon prepared in the transparent packages. Increased NPYR formation in pan-fried bacon was observed with increased nitrite concentrations in the raw product. However, no significant increase in NPYR concentrations was found in microwaved bacon prepared with the higher level of ingoing nitrite. Dedicated to My Family. ii ACKNOWLEDGMENTS The author wishes to express his most sincere thanks and deepest gratitude to his major professor, Dr. B.R. Harte, and co-advisor, Dr. J.I. Gray for their guidance, assistance and encouragement in the course of his graduate study. The author also wishes to thank Dr. J.R. Giacin for his technical advice and support in the preparation of this thesis. The author is grateful to Dr. A.M. Booren, for his assistance to process bacon samples, and Dr. M.A. Uebersax, for his generosity lending the Luxtron Probe System. Sincere thanks and appreciation are given to his laboratory colleagues, Ms R. Crackel, N. Engeseth, E. Gomma, S. Lai and V. Vega for their help and friendship. Last but not least, the author wishes to express deepest appreciation to his family. iii TABLE OF CONTENTS PAGE LIST OF TABLES ................................. ......... vi LIST'OF FIGURES .............. ............ ............... Vii INTRODUCTION ... ..... ... ...... ..... ..... .. ............... 1 LITERATURE REVIEW A Trend to Microwave Foods ...... ..................... 4 Mechanism of Microwave Heating .............. ......... 6 Cooking in A Microwave Oven ................. ......... 11 Parameters Affecting Microwave Heating ............... 13 New Packages for Microwave Foods ........ ............. 15 Safety Concerns of Heat Enhancing Materials .......... 17 Migration ......................... ........... ........ 18 N-Nitrosamines in Foods .............................. 21 Chemisrty of Formation ................. ............ 21 Formation of NDMA and NPYR in Bacon ..... ........... 23 Toxicalogical Concerns of N-Nitrosamines ........... 27 Factors Affecting N-Nitrosamine Formation ............ 28 Cooking Methods .................................... 28 Heating Temperature and Time ....................... 28 Concentration of Residual Nitrite .. ................ 29 Storage of Pork Bellies ............ ..... ........... 30 Thickness of Slices ................................ 3O Lipophilic Inhibitors ................... ........... 31 Fatty Acid Composition of Adipose Tissue ........... 32 smOke 0......0.0.0.0.........OOOOOOOOOOOOOO000...... 33 iv MATERIALS AND METHODS Susceptor Material . ........ ... ....................... 34 Preparation of Bacon ................................. 34 Determination of Ingoing Nitrite Concentration ....... 35 Determination of Power Output of Microwave Oven . ..... 37 Determination of the "Hot Spots" in Microwave Oven ... 38 Thermal Processing of Bacon ................ .......... 39 Determination of Temperatures during Heating ......... 41 Determination N-nitrosamines in Cooked Bacon ......... 41 Statistical Analysis of Data .................. ....... 44 RESULTS AND DISCUSSION Temperature Profile of Microwave Cooked Bacon ........ 45 Evaluation of Degree of Doneness of Bacon ............ 50 Formation of N-Nitrosodimethylamine in Cooked Bacon .. 50 Formation of N-Nitrosopyrrolidine in Cooked Bacon .... 59 Effect of Weight Loss on Formation of N-Nitrosamines . 66 Correlation of N-nitrosamines Formation and Temperature -Time History ................................. ..... 68 Effect of Nitrite Concentrations on N-nitrosamines Formation ........................................... 71 Statistical Analysis ................................. 72 CONCLUSION .............................................. 78 BIBLIOGRAPHY ............................................ 81 LISTS OF TABLES Table Page 1. Microwave Food Features ........................ ..... 5 2. The fundamental differences between conventional and microwave cooking ......................... ........ .. 12 3. Surveys of food for N-nitrosamines .................. 22 4. N-Nitrosamine concentrations in cooked bacon processed in the MSU Meat Lab prepared by using three different cooking methods ..................................... 58 N-Nitrosamine concentrations in cooked bacon purchased from local retail store prepared using three different cooking methods ..................................... 60 N-Nitrosamine concentrations in cooked bacon processed in the MSU Meat Lab. using two nitrite concentrations and prepared using three different cooking methods .. 73 Boferroni t-tests for the formation of NPYR in cooked bacon using different cooking methods and nitrite concentrations in curing brine .......... ..... ....... 77 vi LISTS OF FIGURES Figure Page 1. Rotation of dipole molecules due to changing field .. 9 2. Heat generated by collision of moving ions in the 10. 11. electric field ...................................... 10 Reaction of primary, secondary and tertiary amines with nitrous acid ............................... ......... 24 Possible pathway for the formation of NPYR in bacon . 26 Temperature profile of empty susceptor and transparent materials heated in a 700 watts microwave oven ...... 46 Temperature profile of susceptors either empty or loaded with bacon heated in a 700W microwave oven ... 47 Temperature profile of transparent packages either empty of loaded with bacon heated in a 700W microwave oven ................................................ 49 Bacon prepared using the susceptor packages in a 700W microwave oven ...................................... 51 Bacon prepared using the transparent packages in a 700W microwave oven ............................ ..... 53 Bacon prepared using three different cooking methods to the same degree of doneness ...................... 55 Concentrations of N-nitrosamines in bacon processed in the MSU Meat Lab. and prepared using three different cooking methods .................... ....... 62 vii 12. 13. 14. 15. 16. 17. Concentrations of N-nitrosamines in bacon purchased from a local retail store and prepared using three different cooking methods ...................... ..... 64 Temperature profile of bacon prepared using three different cooking methods to the same degree of doneness ..... ......... ................... ........... 65 Relationship between formation of NDMA and weight loss using microwave in-package cooking methods .......... 67 Relationship between formation of NPYR and weight loss using microwave in-package cooking methods . ........ 69 Correlation of the formation of NPYR and the Temperature-Time History using three different cooking methods to the same degree of doneness .............. 70 Concentrations of N-nitrosamines in bacon processed using two nitrite concentrations in the curing brine and prepared using three different cooking methods .. 74 viii INTRODUCTION Convenience in food.preparation makes the microwave oven a necessary component for most households. A significant time- savings can be achieved with microwave "in-package" cooking. With penetration of microwave ovens in the United States approximately 80%, and retail sales.of:microwave foods several billion dollars, the development of new microwave foods has become an increasingly important Research and Development focus for many food companies (Anonymous, 1988). Insufficient crisping and browning have been the most common negatives associated with microwave cooking (Martin, 1988). Packaging techniques which enhance heating to cause dehydration and browning on the surface of products during microwave cooking, are currently being utilized. Energy absorbing packaging materials, called susceptors, utilize controlled thicknesses of metal, vacuum deposited onto paper, paperboard or film carriers. The metal droplets absorb microwave energy and generate localized heat (Perry, 1986; Dilberakis, 1987; Martin, 1988). Temperatures on the surface of the susceptor have been observed in excess of 200°C (Rosenkranz, 1987; Denford, 1988; labuza, 1988). At these temperatures (>200°C) , several safety concerns have been declared including increased migration of package components into the food product being heated (Bishop, 1987; Denford, 1988; Labuza, 1988; Mitchell, 1988; Rice, 1988b). In one study 2 of a tray-type susceptor (typical for pizza products) it was found that some species migrated close to 100% after 5 - 6 min of microwaving (Mitchell, 1988). It is speculated also, that formation of toxic components such as N-nitrosamines, may result from cooking of bacon in the microwave in contact with susceptors, because of the high temperatures associated with susceptor cooking. Over the past two decades, the amount of N-nitrosamines in foods have come under close scrutiny due to their potential carcinogenic activity in animals. Many Nenitroso compounds have been shown to induce carcinogenic response in a variety of test animals (Hotchkiss, 1987b). N-Nitrosamines have been discovered in a number of food products, especially cured meats, e.g. bacon (Hotchkiss, 1987b). The formation of N- nitroso compounds in cooked bacon is affected by many factors such as: cooking methods (Pensabene et a1., 1974; Bharucha et a1., 1979), cooking time and temperature (Pensabene et a1., 1974; Wasserman et a1., 1978), lean-to-adipose tissue ratio (Fiddle et a1., 1974), lipophilic inhibitors (Sen et a1., 1976; Fiddler et a1., 1978), storage period (Pensabene et a1., 1980), concentrations of residual nitrite (Sen et a1., 1974), thickness of slices (Theiler, a1., 1981) and smoking (Theiler et a1., 1984). Use of susceptors in microwave "in-package" cooking may increase the temperature at the surface of the bacon sample while reducing cooking time (compared to frying), both of 3 factors which may affect the development of N-nitrosamines. The objectives of this study were: 1. To measure the temperature of the product and package surface as a function of microwave cooking in susceptor and conventional (transparent) packages. 2. To develop a correlation between product temperature and formation of N-nitrosamines in microwave package cooking and frying. 3. To determine the effect of residual nitrite on the development of N-nitrosamines as a result of frying and microwave cooking. LITERATURE REVIEW A Trend to Microwave Foods Social and demographic changes in the United State have created a significant increase in the demand for microwavable foods (Wright, 1983). The total number of households in the U. S. is increasing rapidly, while the number of persons per household is decreasing. In addition, more than half of all women between the age of 18 and 50 are in the work force (Martin, 1988). Thus, there is less time for preparation of the traditional meal and more disposable income. Convenience becomes a necessity in meal preparation, due to the lack of desire and/or time to cook (Wright, 1983; Martin, 1988). Changes in lifestyle and user demographics have combined to create an explosion in the marketplace for microwavable foods (Sheridan, 1987). Convenience has been the key element in the success of microwavable foods (Martin, 1988). With approximately 80% penetration, the microwave oven has become one of the more common appliances in the‘U.S. household. About $3.4 billion will be spent in the microwavable food market segment by 1991 (Diberakis, 1987; Huang, 1987). Forty three million servings of food are prepared in the microwave daily, and there has been a 91% increase in microwave oven ownership among young singles and retired people (Sheridan, 1987). Typically, microwave oven owners are younger people between the age of 25 and 35 with total annual incomes over $30,000 5 (Martin, 1988). These people want the total convenience of opening a packaged frozen dinner, putting it directly in the microwave oven, eating it at the table and then throwing it away (Martin, 1988). It is not surprising that the popularity of microwave ovens and higher quality convenience foods has triggered an expanding demand for microwavable products (Wright, 1983). Food processors have also accelerated the usage of microwave ovens by designing products and packages that use microwave heating/cooking to provide high quality products (Perry, 1986). Since there are unlimited opportunities to differentiate. food. products 'using’ current :microwave food packaging, food manufacturers are designing convenient, high quality and time efficiency microwavable foods (Sheridan, 1987). Microwavable foods have many convenience features (Table 1). Table 1. Satisfactory Features of Microwave Cooked Food Convenient Easy Heatable No mess Reheatable No fuss Saves time Quick Simple Innovative 6 The major advantages of microwave heating versus conventional cooking are: s eed, food prepared in the microwave ovens usually take only one quarter as much time, or less required, compared to conventional heating; uniformity of heating, since microwave energy penetrates food more efficiently than by conduction in the conventional oven, temperature distribution is more uniform and overheating may be avoided; high product ggality, quality can be improved by reducing or eliminating case hardening; selective heatin , microwave couples with selective materials which are absorptive of the energy, and which can lead to a greater heating efficiency in multicomponent food systems (Schiffmann, 1986). Change in lifestyles is the primary factor in the U.S. behind the success of microwavable foods (Martin, 1988). In the future, there will be more single households, and less time to cook and prepare meals (Sheridan, 1987). Many more products will be packaged for microwave preparation because consumers appear to want them. Food processors and packagers are actively striving to participate in microwave market expansion. In addition, new microwave packaging technologies are emerging to provide better service, and more convenience features to satisfy the demand (Beck, 1989). Mechanism of Microwave Heating Microwave heating of foods is accomplished by directly transferring energy into foods using high frequency radiation. 7 The frequency agitates dipole molecules within products, and gives up energy as a result of temperature increase through molecule friction (Mudgett, 1986; Ohlsson, 1986; Diberakis et a1., 1987). The most common dipole component in foods is water; Many foods contain large amounts of water, between 50 - 90 % (Ohlsson, 1986). Because of the dipole character of water, the microwave essentially heats products by activating the electrical charges of water molecules. These charges are arranged asymmetrically which is particularly active shape in a microwave field (Sacharow, 1988). Consequently, food products exhibit heating because of the excitation of water due to oscillation within the microwave field (Perry, 1986). Accordingly, microwaves are able to heat food without heating the surrounding environment as in a conventional oven (Bohrer, 1987; Sacharow, 1988). In. a microwave oven, the magnetron sends out electromagnetic waves into the oven cavity, which then bounce around the oven cavity until they are absorbed. The frequency inside the oven cavity results in an oscillating electrical and magnetic field. The.electrica1 and magnetic fields coexist and are oriented in perpendicular planes. These fields are constantly changing, but there are certain configurations of energy that exist at any single point in time (Perry, 1986). Thus, materials with molecular dipoles, mobile ions or mobile electrons are affected by the electric field. The dipoles and ions try' to realign. themselves to ‘the rapidly changing 8 electrical field, the motion created by these molecules causes the heating observed in a microwave oven (Perry, 1986). There are two main mechanisms, by which microwaves produce heat within a microwave-irradiated.product. These are "molecule-friction" resulting from the dipole rotation in a polar solvent and conductive migration of dissolved ions (Mudgett, 1986). The predominant heating mechanism is due to the rotation of dipole molecules as illustrated in Figure 1. In food systems, there are many dipole molecules, though water being the most common dipole material. Under normal conditions, polar molecules are randomly oriented. In the presence of an electric field, the polar molecules line up with the field. As the magnetron generates an oscillating magnetic and electric field (Perry, 1986), molecules will align themselves with the changing electric field. Heat is generated as a result of friction due to the rotation of molecules (Decareau, et a1., 1987). Heat is also produced due to the occurrence of ionic polarization. Conductive migration of ions in solution is induced in response to an electric field (Mudgett, 1986; Derakis et a1., 1987). The moving ions contribute to the kinetic energy by means of the interaction with the electric field, this energy is then converted to heat when the ions collide‘with.others (Fig. 2) (Decareau, et a1., 1987). Though, heat is created predominately through the rotation of dipole molecules, intensified heating will occur if ionic materials \\ - + Fig. 1. Rotation of dipole molecules due to changing field (Decareau, 1987). 10 n 4. I n V V i‘ Fig. 2. Heat generated by collision of moving ions in the electric field (Decareau, 1987). 11 (salts) are dissolved in the aqueous phase (Perry, 1986). Cooking in A Microwave Oven Conventional ovens heat foods from outside in, the same is true in microwave ovens (Perry, 1986; Martin, 1988). An advantage of the microwave cook is that penetration depth is much greater than that experienced in conventional ovens (Perry, 1986) . The essential differences between microwave and conventional cooking is illustrated.in'Table 2 (Bohrer, 1987). In conventional ovens, heated air in the oven gradually increases the temperature of food, which dries food out and aids in browning, crisping and driving moisture away from the surface (Martin, 1988). One further advantage of cooking in a conventional oven is the capability of preparing several food items together (Mudgett, 1989) . Microwave food processes involve transferring energy through the surface, and reaching the interior of food, instead of by conduction (Perry, 1986; Martin, 1988). Typically, the microwave thermal process provides deeper energy penetration, and heats food more efficiently. Mudgett (1988) demonstrated. that. conventional. heating' methods of transfer thermal energy from the product surface to the center are 10 - 20 times more slower, compared to microwave cooking. However, lack of hot air associated reduces browning and crisping, because there is little moisture evaporation at the surface of the food during heating (Martin, 1988). A 12 Table 2. The Fundamental Differences between Conventional and Microwave Cooking. Conventional Oven Microwave Oven Hot air convection Polar molecules excitation Air hotter than food Air cooler than food Heat conducted from surface Microwaves penetrate food Conduction differences Absorption differences relatively small relatively large Surface dehydration before Surface and interior interior dehydrate Surface browning before bulk Surface browning after bulk dehydration dehydration Bohrer, 1987. 13 shortcoming of the microwave oven is its inability to cause browning and crisping (Perry, 1986). The lack of browning and crisping is attributed to low surrounding air temperature at the food surface, and high humidity conditions (Sacharow, 1988) . During the processing of biological materials at medium to high temperatures, extensive mass (specifically moisture) will be transferred from center to surface, and some of it may be temperature-dependent (Ofoli, 1986). Cooling losses due to moisture evaporation at the surface is a major negative factor effecting crisping and browning (Mudgett, 1989). Parameters Affecting Microwave Heating Product size and shape, water, salt and fat content of the food, as well as product density and depth in the container influence the efficiency of coupling the food to the microwave environment (Drennan, 1987). Water content is usually the major attribute which influences the absorption of microwave energy. The more free water present, the higher is its dielectric loss factor, the more efficient is the heating (Schiffmann, 1986). Geometric properties also play an important role in microwaving. The more regular the shape of the food product, the more uniform heating can be obtained (Schiffmann, 1986). Sharp edges and corners tend to overheat, and sometimes cause arcing (Martin, 1988). When microwave energy passes through a curved interface, such as food with cylindrical or spherical geometry, the microwaves will 14 concentrate toward the center of the food, just as light does in a lens. This results in.a high temperature in the center of the food (Ohlsson, 1986). The fat content.also affects heating efficiency during microwaving. Microwave energy absorption by fat or cooking oil is very slow compared to water. However, fat is still heated to a high temperature (in excess of 100°C) , and sometimes can be overheated due to a lower specific heat (Ohlsson, 1986) . The specific heat is a physical index which describes the measurement of the temperature change in a material during thermal processing. When a certain amount of energy is provided to materials with the same weight, the one with lower specific heat will exhibit a greater temperature increase. Oil and fat are heated considerably faster than water because of their far lower specific heat, 2.0 joule /g °C of oil/fat to 4.2 of water (Schiffmann, 1986). Therefore, oil/fat displays twice the increase in temperature from the same quantity of energy, compared to water. Oil/fat demonstrates not only a greater temperature increase, but better energy maintenance. Materials having low boiling point, e.g. water, will consume energy to change their physical form (from liquid to vapor) at low temperature. However, many oil/fat systems do not boil until at least 200°C. More energy is maintained in an oil/fat product instead of lost due to vaporization. During microwaving, the energy absorbed by an oil/fat product may be transferred to the surroundings by conduction. Fats and oils 15 act thermodynamically as a "heat source", and release this energy to adjacent substances, because of the lower specific heat.and.higher boiling temperature (Connerton et a1., 1989). In designing microwave ovens, parameters such as frequency and power output, are primary factors affecting the heating mechanism. Fundamentally, frequency influences the penetration depth and heat response in objects (Schiffmann, 1986; Diberakis et a1., 1987). Two frequencies are currently employed in microwave ovens, these are 915 and 2450 MHz. A frequency of 2450 MHz, which is utilized in household microwave ovens, is more effective in the generation of heat. However, 915 MHz produces a greater penetration depth (Decareau, 1985). The microwave power level also contributes to the time efficiency. The higher the power output, the faster will be the heating for a given mass (Schiffmann, 1986). New Packages for Microwave Foods Currently, several different packaging systems are employed for microwave food products. These include: transparent, absorbing (which are regarded as susceptors) , shielding, and field modifying substances (Perry, 1986). Of these, transparent and absorbing based packages predominate. Susceptors were essentially developed to overcome limitations associated with crisping and browning. In order to achieve significant browning and cuisping reactions, food surfaces 16 must be heated above 300°F (Anonymous, 1988; Sacharow, 1988) . However, temperatures common to microwaving are less than 212°F (100°C) , because of the boiling point of water (Ohlsson, 1986; Martin, 1988) . Temperatures on the surface of susceptors are often in excess of 450°F (Breder, 1988; Labuza, 1988; Larson, 1988) . At these temperatures, rapid dehydration on the surface of the food can be accomplished, resulting in crisping and browning. Susceptors are constructed of a controlled thickness of metal which is usually vacuum deposited onto a plastic carrier, and adhesively laminated to a paper and/or paperboard backing (Huang, 1987; Martin, 1988). The metal layer provides the heating response (Diberakis, 1987) . Molecules in the metal absorb the microwave energy and become agitated. The energy absorbed by the susceptor becomes a source of radiant heat, which reemits to the food surface and promotes crisping and/or browning (Diberakis, 1987; Huang, 1987). The plastic carrier functions as a support surface for the metallization, and protects the metal. The film also provides an approval food contact surface (Diberakis, 1987) . In susceptors, crystallized PET (polyethylene terephthalate) is utilized predominately as the film substrate (Sheridan, 1987) . Paper or paperboard substrates contribute dimensional stability during microwaving (Sacharow, 1988). Susceptors increase the heat to the food by converting microwave energy, to a secondary heat source (Diberakis, 17 1987). A susceptor is usually attached to the cooking vessel in an area where crisping, browning and heat are demanded (Martin, 1988). Aluminum has been the prevalent metal used, because its molecular composition allows it to be heated to very high temperatures and the metallizing technology has been readily available (Martin, 1988). The key to successful heat generation using susceptors is the deposition of tiny droplets of metal onto the substrate surface. The frequency absorbing material must be isolated from one another on the surface of the film. If the film was completely metallized, the metal would act as a microwave shield, and.no heat would be produced (Labuza, 1988). In 1986, approximately 83 million square feet of susceptor materials were consumed in frozen foods such as pizza, fries and. waffles (Rosenkranz et a1., 1987). In general, packaging of microwave foods must be tailored to the surface browning/crisping, interior cooking, and flavor/aroma needs of the particular food item under consideration (Huang, 1988). Safety Concerns of Heat Enhancing Materials Safety concerns have been raised relative to the high temperature process associated.with susceptor based packages. Rapid microwave heating rates may cause rupture, overexpansion, or explosion of bakery products such as jelly doughnuts (Mudgett, 1989). Also, of particular concern is the potential migration of packaging components into food. In 18 addition, at temperatures in excess of 450°F (+200°C) , physico- chemical changes in the food may occur, and result in the formation of hazardous compounds. These hazardous substances include N-nitrosamines in bacon and mutagenic imidazoquinoline compounds in cooked meat (Bharucha et a1., 1979; Bishop et a1., 1982; Bjeldanes et al., 1983; Bieber, et a1., 1984; Felton et a1., 1984; Startin et a1., 1985). M'gration Migration of low-molecular-weight substances from packaging materials into foodstuffs is influenced by the properties of the food components and interaction between the package and food. Migration rates can be affected by many properties of the food, including the fat content, pH value and alcohol content (Bieber et a1., 1984). The mass transport process depends on concentration of migrants, exposure time, system temperature, product affinity to migrants, i.e. solubility and contact surface area (Bieber et a1., 1984). Heat intensifies the migration problem. At high temperatures, breakdown of packaging materials may occur, and result in acceleration in the :migration rate. Moreovery increased migration not only increases the toxicological significance, but also decreases product quality (Bieber et a1, 1984) . Enhanced migration is induced when using susceptors to provide the localized heating of food. At temperatures of 400-500°F, migration of polymer oligomers and additives, 19 polymer decomposition products, adhesives and components of adhesives may occur (Breder, 1988). Bishop (1987) illustrated that for food being heated in plastic containers plasticizers were released from the plastic into the food. The heating of oils or fatty-type foods with microwave energy increases the rate of migration of plasticizers out of plastic film wraps. Increased temperatures and aging of polymer resins also tend to enhance not only the migration of plasticizers, but the migration of monomers and other additives (Bishop, 1987). Typically, the highest temperature specified for any packaging film by the Food and Drug Administration (FDA) is 275°F, however, microwave susceptors often heat at 400-500°F (Labuza, 1988; Mitchell, 1988). At temperatures in excess of 500°F, there is no functional barrier between the food and the susceptor packaging components (Mitchell, 1988). A close examination of microwaved packages shows "clear evidence" of cracking and melting of the polyester film and browning of the paperboard.(Mitchell, 1988). A simple dye test revealed that the cracking went below the surface and penetrated into the paper and adhesives layers. The migration of PET residues into corn oil illustrated that extent of migration increased with the heat experienced. With the tray- type susceptors used in pizza products, migration levels were doubled compared to virgin PET after'5 to 6 min of microwaving (Mitchell, 1988). Recently, the FDA has expressed safety concerns on the 20 degradation problem with adhesives, polymers, paper and other components of susceptor microwave packages. The major concerns of the FDA were as follows (Hollified, 1988): 1. Breakdown of packaging material (packaging materials begin to crack during microwaving). 2. Chemical migration from the food contact surface increases during microwaving. 3. Does food product contact layer provides sufficient protection of the food from potential migrants? At the extremely high temperatures (450 - 500°F) generated in susceptors by microwave heating, the possibility of migration, ignition of oils and fats, and other possible mishaps have sparked an industry-wide clamor for government- approved testing procedures for microwave foods/packaging (Larson, 1988). 21 N-Nitrosamines in Foods For the past two decades, the concentration in food.of N- nitroso compounds has been monitored carefully, because of their potential carcinogenic activities in animals. The acute toxicity of N-nitrosodimethylamine was investigated in animals in the 1930's. Severe liver failure was found, and the more acutely poisoned individuals died of liver necrosis approximately 7 weeks after exposure (Hotchkiss, 1987b). More than 80% of the 300 N-nitroso compounds tested.have been found to be carcinogenic in relation to one or more animal species (Magee et a1., 1976). N-Nitrosamines have been observed in a number of food products, including cured meat (especially cooked bacon) , dairy products, tobacco and alcoholic beverages (Table 3) (Hotchkiss, 1987b). The N—Nitrosamines in bacon are primarily N- nitrosodimethlyamine (NDMA) and N-nitrosopyrrolidine (NPYR). Many factors affect the concentration of these compounds in cooked bacon, including cooking method, cooking temperature and time, residual nitrite, lipophilic inhibitors, lean-to- adipose ratio, age.of pork bellies, thickness of slices, fatty acid composition, and smoking process (Skrypec et a1., 1985). Chemistry of Formation N-Nitrosamines are primarily formed from the reaction between secondary amines and nitrosating agents, but also can be produced from primary, tertiary and polyamines (Fig. 3) 22 Table 3. Survey of Foods for N-Nitrosamines. Survey and Food No. positive N-Nitroso Range /No. analyzed compounds (pg/kg) Havery et al. (1976), in U.S. Fried bacon 22/22 NPYR 7-139 Variety meats 1/ 6 NPYR 6-NRa Gough et al. (1977), in U.K. b Fried bacon 33/56 NDMA, NPYR ND-200 Canned, cured meats 9/34 NDMA ND- 10 Fish 28/112 NDMA ND- 10 Cheese 10/58 NDMA ND- 15 Cured meats 25/64 NDMA, NDEA ND- 8.6 Kawabata et a1. (1980), in Japan Salt-fermented 6/49 NDMA, NPYR ND- 5 vegetables Beer 27/29 NDMA, NPYR ND- 5 Spiegelhalder et a1. (1980), in Germany Meat product 127/395 NDMA, NPYR 0.5- >5 Cheese 49/209 NDMA 0.5- 5 Beer 142/215 NPYR ND- 68 Sen et a1. (1980), in Canada Cured meats 77/118 NDMA, NDEA ND- 55 NPYR, NPIP Alcoholic beverage 23/50 NDMA ND- 4.9 Osterdahl (1988), in Sweden Fried bacon 68/68 NDMA, NPYR 1.7- 7 6 Smoked pork, fried 20/21 NDMA, NPYR 0.9- 4 0 Smoked fish 55/61 NDMA ND- 1 3 Beer 153/258 NDMA ND- 0 3 Spice mixture 4/4 NDMA, NPYR 0.7- 1 5 NPIP a. NR, not reported b. ND, not detected Hotchkiss, 1987b; Osterdahl, 1988 23 (Gray et a1., 1979). In an acidic environment, nitrite is converted to nitrous acid (HONO) which then reacts with primary, secondary and tertiary amines to form N-nitrosamines (Lijinsky et a1., 1972). When secondary amines are treated with nitrous acid, nitrosation occurs and stable N-nitrosamines are formed (Fig. 3). The first step in the nitrosation of a tertiary amine is similar to the reaction path.for'primary and secondary amines. The reaction is accomplished when the unshared electron pairs with the unprotonated amines and reacts with a nitrosating agent (nitrous anhydride), forming the nitrosoammonium ion (Fig. 3). These ions undergo cis-elimination of the nitroxyl ion to form immonium ions which are hydrolyzed to carboxyl ions. Thus, the secondary amine is nitrosated to the corresponding N-nitrosamines (Fiddler, et a1., 1972). Formation of NDMA and NPYR in Bacon N-nitrosamines have been frequently detected in cooked bacon, including NDMA, NPYR, Nenitrosodiethylamine (NDEA), NPYR and Nhnitrosopiperidine (NPIP). However, the major N- nitrosamine present in bacon is NPYR. NDMA has been shown to be more carcinogenic than NPYR (Magee et a1., 1976)., and is found consistently in bacon. Up to 80% of NDMA is lost in the vapor during preparation (Gray et a1., 1977). The formation of NDMA in bacon is not clearly understood. However, results from several model studies have Primary Amines RNHZ 1° aliphatic amine Secondary Amines RR‘NH or + ArRNH HNo2 2° aliphatic or aromatic amines Tertiary Amines R1 RNCHZRZ + 3° aliphatic amines + H1102 —-+ RNENN HNo2 24 ——-» N2 -+ ROH + =R diazonium nitrogen alcohol alkene ion RR1N=NOH RR‘NN=O —-> or —_. or + H20 ArR1N=NOH ArNN=O N-nitrosamines 1 1 Re R —-> RNCHZRZ —-> RN=CHR2 + HNo+ N e H . . . O immonium nitroxyl ion nitrosammonium ion H20 1 HNo2 RR‘ =o «— RR‘NH + ch=o H 2° aliphatic aldehyde amine Fig. 3. Reactions of primary, secondary and tertiary amines with nitrous acid (Lee, 1981). 25 indicated that various compounds, including lecithin, sarcosine, and tri- and di-methylamines can form NDMA on reaction.‘with. nitrite (Fiddler' et .al., 1972). Compounds containing sarcosine and choline were proposed to be the most probable amine precursors of NDMA (Gray, et a1., 1978). However, the concentration of sarcosine and choline in pork bellies has not been estimated. The consistent occurrence of NPYR in fried.bacon.has.been reported in many studies (Havery et a1., 1976; Gough et a1., 1977; Spiegelhalder et a1., 1980; Sen et a1., 1980; Osterdahl, 1988). Studies of food systems have implicated several compounds including proline (PRO), pyrrolidine (PYR), collagen, putrescine and spermidine as potential precursors of NPYR, with PRO as the most likely candidate (Gray, 1976). Although the formation of NPYR from PRO has not thoroughly understood, two pathways have been proposed (Gray, 1976; Bharucha et a1., 1979). The jpossible jpathways for' NPYR formation in bacon are illustrated in figure 4.: in the first pathway, PRO is initially nitrosated to N-nitrosoproline (NPRO), NPRO then undergoes thermal decarboxylation to yield NPYR. In the second pathway, the initial thermal decarboxylation of PRO to pyrrolidine (PYR), is followed by nitrosation to NPYR. Since decarboxylation of NPR0 to NPYR occurs at a much lower temperature than from PRO to PYR, the initial pathway seems the more favorable (Bharucha et a1., 1979; Lee et al., 1983b). 26 9m? .00.: .coomo E m>dz Lo cozEmtoe mf toe >ms>£ma oEBwoa .v .mE m_Z_.._Omn_OwOm._._Z..Z 0 n2 MZ_O_:_Omm>n_ Z OwOmCZIZ IOOO \ /_ wZ_n_Omn_ .. z mz_o_._0mm>n_ Iooo_\ J - , I 02. _\ J 1 :ooo- coszzo>o coszEmmo m2_0wwm._.3n_ 27 Toxicological Concerns of N-Nitrosamines About 300 N-nitroso compounds have been tested, and more than 80% have been shown to have carcinogenic activity in laboratory animals (National Academy of Science, 1981) . However, the major safety concern of N-nitroso compounds to human is the chronic exposure, instead of the instantaneous response. The environment/food exposure rates of N-nitroso compounds is less than the amount required to produce an acute toxic effect in the short term (Hotchkiss, 1987b). However, the concentrations are high enough to cause tumors in experimental animals through long term exposure (Magee et al. , 1976; Sen et a1., 1980). It is generally believed that N-nitroso compounds must be metabolically activated to express carcinogenic activity (Archer, 1982) . The metabolic activation of N-nitrosamines involves enzymatic hydroxylation of an a carbon atom to the N- nitrosoamine nitrogen. Therefore, the alpha position of the N- nitrosamines has been associated with the carcinogenic action of these compounds (Magee et a1., 1976). Animal experiments indicate that the effect of the N- nitrosamine in chronic exposure is cause liver necrosis and/or hemorrhaging, and the carcinogenesis of N-nitrosamines has been shown to be organ specific (Hotchkiss, 1987b) . N- Nitrosodimethylamine and N-nitrosopyrrol idine, the most widely occurring N-nitrosamines in foods, cause mostly liver tumors and occasionally kidney tumors in animals (Magee et a1., 28 1976). Factors Affecting N-Nitrosamine Formation Factors influencing the formation of N-nitrosamines in bacon are well documented (Skrypec et a1., 1985). Studies outlining contribution of the different factors are discussed below. Cooking Methods Pan frying has been shown to produce greater amounts of N-nitrosamine in bacon, compared to broiling and grilling (Pensabene et a1., 1974; Wasserman et a1., 1978; Bharucha et a1., 1979). A.high internal temperature contributes.to‘greater N-nitrosamines in cooked bacon (Wasserman et a1., 1978). Similarly, draining of the cook-out-fat during grilling lowered the internal temperatures, and resulted in less N- nitrosamine formation in bacon (Bharucha et a1., 1979). Heating Temperature and Time Temperature and time of‘cooking'play'an important role in the formation of N-nitrosamine. Only 10% conversion to NPYR was found at 100°C compared to that produced at 180°C (Pensabene et a1., 1974). The maximum NPYR formation in bacon was observed when bacon was fried at 182°C (350°F) for 12 min (Bharucha et a1., 1979). Generally, the N-nitrosamine content in cooked bacon begins to increase after 4 min, and reaches 29 maximum at about 12 min, and then declines due to volatilization (Pensabene et al., 1974). Pensabene et a1. (1974) also found that the optimum temperature for N- nitrosamine formation was 185°C, which is near normal pan frying temperatures. During frying, the internal temperature of the lean tissue reached a maximum of 110°C (Coleman, 1978) . However, after the majority of the water has vaporized, the temperature of rashers can reach 180°C (Coleman, 1978) . Therefore, it was proposed that the majority of N-nitrosamines are produced after the bulk of water in bacon has volatilized (Pensabene et a1., 1974; Coleman, 1978; Bharucha et a1., 1979). Concentration of Residual Nitrite The role of nitrite in cured meat functions to produce the characteristic cured meat color, texture and flavor, and inhibits the outgrowth of Clostridium botulinum.and.acts as an antioxidant (Hotchkiss et al., 1987a). However, nitrite is also the primary reagent involved in N-nitrosamine formation. The rate of nitrosation is directly proportional to the square of the nitrite concentration (Mirvish, 1975). Sen et al. (1974) reported.that.NDMA and.NPYR.concentrations in fried bacon were highly correlated with the initial nitrite level. Robach et a1. (1980) reported that a reduction in the initial sodium nitrite level from 120 to 80 mg/kg, resulted in significantly lower NPYR concentrations in bacon. Therefore, 30 the lower the residual nitrite the less is the probability of N-nitrosamine formation (Dudley, 1979). Storage of Pork Bellies The length of storage of pork bellies prior to processing affects the final N-nitrosamine formation in cooked bacon. Greater concentrations of NPYR were reported in fried bacon, when aged bellies were used in comparison with fresh pork bellies (Pensabene et a1., 1980). The free proline content has been shown to increase by 50% in whole bellies, and approximately 90% in the adipose tissue after one week of storage at 2°C (Gray et a1., 1977). The increase in free amines during aging due to proteolysis, therefore, produces greater NPYR concentrations after frying (Pensabene et a1., 1980). Thickness of Slices Since the formation of N-nitrosamines is related to product cooking temperature of frying, the thickness of the slices may affect NPYR formation. Theiler et al. (1981a) illustrated that as thickness increased, NPYR concentrations decreased. Thicker slices do not allow as high an internal temperature to be reached during frying, and results in decreased quantities of N-nitrosamines. 31 Lipophilic Inhibitor Bharucha et al. (1979) observed that formation of N- nitrosamines during’ the frying’ of Ibacon occurred. almost entirely in the fat phase by a free radical mechanism rather than an ionic mechanism. A compound which can successfully compete with a secondary amine for a nitrosating species results in the reduction of N—nitrosamines formation, is regarded as a blocking agent (Gray et a1., 1975). Lipophilic inhibitors, function as blocking agents, and serve to trap NO radicals in the fat phase, and block the nitrosation reaction of amines (Bharucha. et a1., 1979). Alpha-tocopherol and ascorbic acid derivatives have been shown to inhibit N- nitrosamine formation by lipophilic reduction (Sen et a1., 1976; Fiddler et a1., 1978; Bharucha et a1., 1980). Reduction in N-nitrosamines by ascorbic acid and its isomer, erythorbic acid (g-erythro-ascorbic acid), was first discussed. by' Mirvish. et al. (1972). Inhibition involves oxidation of the vitamin C to dehydroascorbic acid, and the reduction of nitrous acid to nitric oxide (Mirvish et a1., 1972). Another ascorbate compound, ascorbyl palmitate, also reduces N-nitrosamine formation in bacon by 70-90%, when 500 - 1000 mg/kg is added in the brine (Bharucha et a1., 1980). Although, reduction in N-nitrosamine has been accomplished using these compounds, ascorbates are not completely successful in inhibiting NPYR formation, because of their limited solubility in adipose tissue. 32 Alpha-tocopherol has been reported to inhibit NPYR formation by 90% when used at a level of 500 mg/kg (Gray et a1., 1982). This lipophilic antioxidant reacts with nitrosating agents in a manner similar to ascorbates. However, a-tocopherol has the added benefit of not undergoing C- nitrosation (Mergens et a1., 1979) . a-Tocopherol reduces nitrogen oxide to form nitric oxide, while it undergoes oxidation to quinone (Hotchkiss, 1987b) . An optimum inhibition of nitrosation was obtained using 500 mg/kg of a-tocopherol and 550 mg/kg of ascorbate (Gray et a1., 1982). The combination of a-tocopherol and ascorbate more effectively inhibited.N-nitrosamine formation than either compound alone. This is due to the fact that ascorbate inhibits N-nitrosamine formation.in the aqueoquphas , while.a-tocopherol acts in.the lipid phase. Fatty Acid Composition of Adipose Tissue Formation of the majority of N-nitrosamines appears to be associated primarily with the adipose tissue during frying (Pensabene et a1., 1974; Bharucha et a1., 1979). Increased NDMA and NPYR formation in fried bacon were observed in association with elevated amounts of unsaturated fatty acids in the pork.bellies (Skrypec et a1., 1985). The fat.portion in bacon yields 12 times more NPYR, and 6 times more NDMA than the lean portion during frying (Mottram et a1., 1977). One possible mechanism was illustrated by Walter et a1. (1979). ‘1 2a 33 Unsaturated fatty acids were shown to act as transnitrosating agents in the fat phase. The study demonstrated that unsaturated fatty acids are.capable.of interacting with.nitric oxide (nitrite) to form a-nitroso-nitrite esters with unsaturated double bonds. The a-nitroso-nitrite esters of unsaturated triglycerides can then transnitrosate to secondary amines to produce N-nitrosamines in the adipose tissue. Smoke Cured meats are smoked in order to enhance preservation, and to develop characteristic flavor and color. Pensabene et al. (1983) indicated that some nitrosating species may be generated by nitrogen oxides during the smoking process. Some components of wood smoke, including formaldehydes and nitro- phenols, could possibly be involved in the N-nitrosation reaction (Mandagere, 1986). However, Bharucha et al. (1980) reported that smoked bacon contained less NPYR and/or NDMA compared to unsmoked samples, presumably due to a lowering of pH by the acidic smoke ingredients. The effects of smoking seem to be due to a combination of lowering the pH, and direct C-nitrosation of phenolic compounds. METHODS AND MATERIALS Susceptor Material The susceptor material was obtained in sheet form (60.5 cm x 90.7 cm) from a material manufacturer. The susceptor material was constructed of metallized (aluminum) polyethylene terephthalate (PET), and adhesively laminated to paperboard. The PET surface is the food contact layer. Preparation of Bacon Samples Pork bellies were processed into bacon within 48 hr of slaughter in the Meat Laboratory, Michigan State University. Bellies were stitch pumped to 110% (wt/wt) of their green weight using a brine, to obtain a target concentration of 1.5% sodium chloride, 0.35% sodium tripolyphosphate, 0.5% sucrose, 120 mg/kg sodium nitrite, and 550 mg/kg sodium ascorbate. To study'the.effect.of nitrite concentrations on the formation.of N-nitrosamines, pork bellies were cured using the same brine as described previously, except bellies were pumped to obtain a target concentration of 200 mg/kg sodium nitrite instead of 120 mg/kg. Pumped bellies were placed into polyethylene bags after stitch pumping, and the bellies allowed to equilibrate overnight at 2°C. Bellies were then transferred to an Elec- Trol laboratory smokehouse (Drying System Inc., Chicago, IL), and cooked at 58°C (dry bulb temperature) for 4 hr, followed 34 35 by three hours of cooking at 52°C (dry bulb temperature). The smoked bellies were transferred to a cooler (2°C) and stored overnight.prior'to slicing and packaging (Reddy et al., 1982). After smoking, the slab bacon was sliced into 1/8 in (0.32 cm) thick slices. Bacon was placed onto polystyrene trays, deposited into polyethylene pouches, and then sealed under vacuum. The packaged samples were stored at 2%: for 7 days prior to cooking. Two commercial slabs of bacon, purchased from a local retail store were also used in this study. Samples were sliced (0.32 cm thick), placed onto polystyrene trays and sealed under vacuum in polyethylene pouches immediately after purchase. The raw bacon was stored at a temperature of 2°C for one week prior to cooking. Following cooking, bacon samples were stored in 18 oz polyethylene pouches (Whirl-Pak, NASCO, Ft. Atkinsos, WI). Cooked samples were deposited into separate pouches, closed and placed in a freezer at a temperature of -5°C prior to analyze the cooked bacon samples for N-nitrosamine content. Determination of Residual Nitrite Residual sodium nitrite in the raw bacon was determined using the.AOAijrocedure (AOAC, 1984). A.5 g finely comminuted sample was weighed and mixed with 40 ml (80°C) water to break up the lumps and then transferred into a 500 ml volumetric flask. To the mixture was added enough hot water to bring it toc ,‘1. AA. 36 to ca 300 ml. It was then placed in a steam bath for 2 hr. After cooling to room temperature, the mixture was diluted to 500 ml using distilled water. The solution was filtered using Whatman No. 1 filter paper, and 25 ml of filtrate was transferred to a 50 ml volumetric flask. A 2.5 ml volume of sulfanilamide reagent was added to the filtrate, along with 2.5 ml of NED (N-(l-naphthyl) ethylenediamine) reagent after 5 min. The solution was diluted to 50 ml using distilled water, and the color was allowed to develop for 15 min. Residual nitrite in the raw bacon was determined using a spectrophotometer (Spectronic 2000, Busch & Lomb, Rochester, N.Y.) to measure the absorbancy at 540 nm. A mixture of 45 ml distilled water, 2.5 ml sulfanilamide and 2.5 ml NED reagent was used as a blank to calibrate the optical response of nitrite. A standard curve was used to determine nitrite present in the raw bacon. The amount of residual nitrite was based on comparison of the absorbancy at 540 nm to a standard curve. The standard curve was established using 1, 2, 5, 10, 20, 30 and 40 ml of a 1 ug/ml NaNOz solution. 37 The concentration of residual nitrite in the samples was calculated as: 500 ml Total AMOUNT READ [ FROM ] x [ 25 ml Aliquot ] = NITRITE STANDARD CURVE (mg/kg meat) (#9) 5 g sample Determination of Power Output of Microwave Oven The power output of the microwave oven was calibrated prior to cooking the bacon. The power output was determined three times, and an average reported. The measurement was accomplished at the maximum microwave power setting with a load of 2000i5 g of potable water. The microwave oven was clean and free from grease using paper towels to wipe the cavity wall prior to cooking, and then warmed up by placing a one liter' beaker containing a liter of water in the cavity. The microwave oven was operated on full power (100%) for 10 min. The oven was then allowed to cool to room temperature, and the test repeated within 20 minutes of the oven shut off. Potable water (2000 g) was divided equally into two 1-liter beakers, the initial temperature of‘water was in the range 18 - 22°C (64.5 - 71.5°F). The water was stirred using a glass rod, and the initial temperature in each beaker was measured and recorded. The beakers were placed in the center of the oven in contact with each other. The microwave oven was operated at full power for 2 min and 2 sec (to compensate for the filament 38 warm up time). The water in each beaker was stirred immediately after heating. The temperature was then measured and recorded in each of the beakers. This procedure was repeated two more times, each test was repeated within 20 min of shutoff of the last test. To determine microwave power output in watts the following calibration was used as follows: AT1-+ A T2 POWER (Watt) = 70 { } 2 where, AT 1 2are the temperature rise of the water in the beakers in degrees Celsius, which. is determined. by subtracting' the initial water temperature from the final temperature. Determination of the "Hot Spots" in the Microwave Oven The susceptor microwave material functions by absorbing energy from the magnetron in the microwave oven. The energy inside ‘the oven. cavity’ is. delivered in 'the form. of an oscillating electrical and magnetic field. The field changes at any point in time, therefore, localized heating exists, which creates "Hot" and "Cold" spots (Perry, 1986). A spot in the path of the microwave field, where responding intense energy results in a high temperature, is characterized as a hot spot. Faster heating is obtained when samples are placed on the hot spot. Therefore, determination of hot spots is 39 necessary prior to all microwave induced thermal processes. Susceptor sheets were used to determine the hot spots in the microwave oven (Amana Refrigeration Inc., model RS458P, 700 Watts, Amana, IA). A significant attribute of susceptors is their ability to absorb microwave energy and convert it into heat. In the construction of a susceptor, a controlled thickness of metal is deposited on a plastic carrier, and laminated to a paper/paperboard backing (Huang, 1987; Martin, 1988) . Since the paper backing has only moderate thermal resistance, charred.marks will be observed.as a result of heat from the aluminum particles. These marks are characterized as the hot spots. Determination of the hot spots was accomplished using a sheet of susceptor material cut to match the inside dimensions of the oven floor, and placed directly onto the floor of the oven cavity. The susceptor was then exposed to 5 min of heating at full power. The susceptor sheet was then inspected and the location of the hot spots were recorded. Thermal Processing of Bacon Bacon samples were prepared conventionally in a fry pan and in two different microwave packages, in order to determine the effect of cooking methods on the formation of N- nitrosamines. Frying was accomplished using a preheated teflon-coated electric pan at a thermostat setting of 171%: (340°F) for 3 min on each side (6 min total). Three slices of 40 bacon (80 - 100 g) were fried simultaneously, and a total of 15 slices of bacon were cooked using frying. After frying, the fried bacon samples were placed on paper towels to drain away the cook-out-fat. Microwave "in-package" cooking methods were accomplished using the susceptor and microwave transparent materials in a microwave oven (Amana Refrigeration Inc., model RS458P, 700 watts, Amana, IA). The output power of the microwave oven was calibrated.prior to use, and found.to be 670 watts. IMicrowave transparent packages were commercial products, purchased from a retail store. The microwave transparent trays were made from CPET (allowed the temperature of use no to 450°F), and had dimensions of 15.3 cm x 20.5 cm (W x L). Susceptors were obtained from a material supplier. The susceptor was used by cutting the susceptor sheets into the same dimensions as the bottom area of the transparent tray, and then placing the susceptor onto the bottom of the tray. The bacon samples were cooked in the microwave oven at full power. Three slices of bacon (80 - 100 g) were used in both microwave cooking methods, 15 slices of bacon were used in each microwave cooking. Cook-out-fat was drained off on paper towels. Heating times were varied to achieve a specific "degree of doneness" compared to frying. Cooking times of 2, 3 and 4 min were used for transparent in-package cooking, and 2, 2.5 and 3 min for susceptor in-package cooking. Degree of doneness was based on the browning characteristic of the 41 cooked bacon and "same degree of doneness" determined using a small panel. Determination of Material and Bacon Temperatures during Cooking Using Fluoroptic Probes and Thermocouples A thermocouple system (Omega Engineering Inc., Gardiner, N.Y.) was used to measure the temperature of bacon during frying. Four probes were placed at the interface between the bacon sample and the frying pan. Temperature readings were recorded every 30 seconds. For the bacon prepared in the microwave oven, temperatures on the interface of the packaging material and bacon surface were measured using a Luxtron 755 Multichannel Fluoroptic Thermometer (Luxtron, Mountain View, CA). Two MIW probes were placed onto the surface of the empty microwavable packaging material and/or interface between the samples and the packaging material. Temperatures were measured and recorded every 10 seconds using a data collection computer system. Determination of N-Nitrosamines in Cooked Bacon N-Nitrosamine concentrations in cooked bacon were quantitated using A Dry Column - Thermal Energy Analyzer method (AOAC, 1984). All chemicals were analytical grade and used without further purification. Dichloromethane (CHZClZ) was purchased 42 from Mallinckrodt Inc. (Paris, KY), and redistilled prior to use. N-Pentane was HPLC grade and also purchased from Mallinckrodt. N-Nitrosamine standards were purchased from Thermedics Inc., Woburn, MA.. Celite 545 (Fisher Scientific Co.) was prewashed with CHZCl2 twice, and then dried in a oven (110°C) for 4 hr prior to use. Celite (10 g) was mixed with 10 ml of 6N HCl, and poured into a chromatographic column. Bacon samples were blended in dry ice using a Waring Blender (model 33BL79, New Hartford, CT), and then held at room temperature until all dry ice evaporated. Ten grams of the bacon were transferred into a mortar, and 25 g of anhydrous sodium sulfate (Na2804) and 10 g Celite were added and mixed into the bacon. This mixture was then poured into the column. One ml of the internal standard, N-nitrosoazetidine (NAZET) (0.1 pg / ml CHZClZ) , and 30 g of NaZSO, were deposited on top of the column. Extraction was initiated by adding 100 ml of a N-pentane - dichloromethane mix (95 : 5). When the level of solvent dropped to the top of the NaZSOU 125 ml CHZCl2 was added to the column. The initial 85 ml of wash eluent was discarded, and the remaining eluent was collected into a 125 ml flask. The eluent was transferred into a Kuderna-Danish (K-D) flask (Knote Glass Co.) equipped with a concentrator tube which was attached to a 3-section Snyder distillation column (Knote Glass Co.). The eluent was concentrated to 4 ml using a steam bath, and then concentrated to 1 ml by flushing with nitrogen. 43 An aliquot (5 pl) of the concentrated extract was then injected into a GC-TEA (Varion Aerograph G.C. model 3700 interfaced with a TEA Model 502) . A 2.7 m x 3.2 mm (ID) glass column (Supelco, Bellefonte, CA) packed with 15% Carbowax 20M- TPA was used to separate the N-nitroso compounds. Thermal conditions were set initially at 140°C, and programmed to 180°C at a rate of 7°C / min. The peak area of the extracted N- nitrosamines was calculated using a Hewlett Packard Model 3390A Integrator (Walnut Creek, CA). The analytical efficiency was monitored by determining the recovery of the internal standard (NAZET). An aliquot of 5 pl (0.1 pg NA /ml) standard solution containing seven N- nitroso compounds, including N-nitrosodimethylamine (NDMA) ; N- nitrosodiethylamine (NDEA); N-nitrosodipropylamine (NDPA); N- nitrosodibutylamine (NDBA); N-nitrosopiperidine (NPIP); N- nitrosopyrrolidine (NPYR) and N-nitrosomorpholine (NMOR) , was used for identification and quantitation of N-nitroso compounds in the cooked bacon. Specific N-nitrosamines were identified by comparing their retention time to those of the standard N-nitrosamines, and the amount of N-nitrosamines in the bacon was determined according to their relative response area . 44 The amount of N-nitrosamines formed in cooked bacon was calculated by: Asample CONC. OF STANDARD N-nitrosamine = ( ) x ( ) x 1000 where, A represents the response area of N-nitrosamines sample' in cooked bacon. A represents the response area of N- stendard’ nitrosamines in the working standard. STATISTICAL ANALYSIS Correlation between formation of N-nitrosamine and cooking methods was determined by measuring the NPYR concentration versus Temperature-Time History of the different cooking methods. The Temperature-Time history was determined by integrating the area under the Temperature - Time profile. Statistical comparison of N-nitrosamine data relative to the three cooking methods and residual nitrite levels was accomplished using the Bonferoni t-test (Gill, 1978). RESULTS AND DISCUSSION Temperature Profile of Microwave Cooked Bacon A rapid increase in temperature on the surface of the empty susceptor was obtained during heating in a 700 Watt microwave oven. Temperatures in excess of 100°C (212°F) were observed within 30 sec. A final temperature of 220°C (430°F) was obtained on the susceptor material after microwave heating for 3.5 min (Fig. 5). Less heat. was generated in the transparent package. An elevation in temperature was found on the surface of the empty transparent package during microwaving. However, no temperature greater than 110°C (230°F) was observed after' 6 min. of microwaving (Fig. 5). The susceptor material more efficiently converted microwave energy into heat than did the transparent package (Fig. 5). Lower temperatures were obtained when the susceptor packages were loaded with bacon during heating. A decreased temperature was observed after cooking for 2 min (Fig. 6). The highest temperature recorded was 113°C (235°F) . Higher temperatures were observed with increased cooking times. Final temperatures of 166°C and 211°C (331°F and 412°F) were obtained at 2.5 and 3 min of microwaving, respectively (Fig. 6). However, no temperature was observed greater than those found on the empty susceptor for the same time period (Fig. 6). 45 AOV NEDIP z 1z