Establishing Threshold Levels of Nitrite Causing Pinking of Cooked Meat

Transcription

1 Utah State University All Graduate Theses and Dissertations Graduate Studies 1998 Establishing Threshold Levels of Nitrite Causing Pinking of Cooked Meat Kevin M. Heaton Utah State University Follow this and additional works at: Part of the Food Chemistry Commons, and the Food Processing Commons Recommended Citation Heaton, Kevin M., "Establishing Threshold Levels of Nitrite Causing Pinking of Cooked Meat" (1998). All Graduate Theses and Dissertations This Thesis is brought to you for free and open access by the Graduate Studies at It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of For more information, please contact

2 ESTABLISHING THRESHOLD LEVELS OF NITRJTE CAUSING PINKING OF COOKED ~AT by Kevin M. Heaton A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Ill Nutrition and Food Sciences Approved: UTAH STATE UNIVERSITY Logan, Utah 1998

3 11 ABSTRACT Establishing Threshold Levels ofnitrite Causing Pinking of Cooked Meat by Kevin M. Heaton, Master of Science Utah State University, 1998 Major Professor: Dr. Daren P. Cornforth Department: Nutrition and Food Sciences Sporadic problems with pink color defect, or pinking, has occurred in cooked meat products for decades. Pink color can be due to the presence of undenatured myoglobin, denatured globin hemochromes, or nitrosylhemochrome. This research documented the level of added nitrite that produced nitrosylhemochrome in processed meat rolls from fabricated beef round, pork shoulder, turkey breast, and chicken breast. For each meat type, preliminary studies were conducted to narrow the range at which added nitrite caused pinking. Subsequently, the nitrite levels were increased incrementally by 1-ppm, and pink color was measured by trained panelists and by a Hunter color meter. Nitrosylhemochrome content was determined by acetone extraction.

4 111 Panel and instrumental measurements identified pink color in beef samples formulated with 14-ppm nitrite; nitrosohemochrome extracts detected pigment at 12- ppm. Nitrite levels that caused pinking in pork shoulder were much lower than in beef. Panelists identified pink color at 4-ppm nitrite, and Hunter color meter values showed increased redness at 6-ppm. Pigment extraction detected nitrosylhemochrome at 4- ppm added nitrite. The trained panel and Hunter color meter detected pink color in turkey breast at 2-ppm added nitrite; nitrosohemochrome extraction detected pink pigment at 3-ppm added nitrite. In chicken breast, pink color was detected visually and instrumentally at 1-ppm added nitrite. Pigment extraction detected nitrosylhemochrome at 2-ppm added nitrite. Lower levels of nitrite (1-3-ppm) caused pinking in light-colored meats (turkey and chicken breast, meats with total pigment between 19-ppm and 27-ppm). Higher levels of nitrite (5-14-ppm) caused pink color defect in dark pigmented meat (beef round and pork shoulder, meats with total pigment levels between 56-ppm and 147-ppm). Regression analysis was used to relate total pigment and the minimum level of nitrite causing pinking. The minimum nitrite level causing pinking was the lowest level of nitrite at which the trained panel, acetone extraction, and instrumental results detected pink color or nitrosyl pigment. The formula obtained from the model was as follows: Y = 0.092X , where "Y" is the minimum level of added nitrite to cause pinking and "X" is the total pigment of the meat. This formula can be used to estimate the level of nitrite that can be expected to cause pinking in a wide range of pigmented meats. (83 pages)

5 IV ACKNOWLEDGMENTS I would like to express my appreciation to my major professor, Dr. Daren Cornforth. He has spent countless hours studying the topic of meat color. His expertise in meat color greatly facilitated this research. Daren was also very patient waiting for me to complete this thesis and was a good friend through it all. Also, members of my committee, Dr. Charles Carpenter and Dr. Deloy Hendricks, deserve my thanks. Not only did they participate on several of my trained panels and gave helpful suggestions, but they always had a good hunting story to tell. I would like to thank the sponsors of my research assistantship, the Utah State University Center for Excellence in Meat Processing, funded by the state of Utah. The completion of this project would not have been possible without the help and support of my family, friends, and, especially, my wife, Melanie. Another motivating factor in completion of my thesis was the fact that someday I would be out of school, and I could use the knowledge in my career. Thank you. Kevin M. Heaton

6 v CONTENTS Page ABSTRACT ii ACKNOWLEDGMENTS iv LIST OF TABLES vii LIST OF FIGURES xiii INTRODUCTION OBJECTIVES LITERATURE REVIEW Hemoglobin and Myoglobin Meat Color Chemistry Cured Meat Color Cured Meat Attributes MATERIALS AND METHODS Experiment Design Sample Preparation Analysis of Samples RESULTS AND DISCUSSION Beef Round Pork Shoulder Turkey Breast Chicken Breast Total Pigment Extraction CONCLUSION REFERENCES APPENDICES

7 Vl Appendix #1-BeefData 0 Appendix #2-Pork Data 0 Appendix #3-Turkey Data 0 Appendix #4-Chicken Data

8 Vll LIST OF TABLES Table Page 1. Pigments found in fresh, cured and cooked meats, mode of formation, state of iron, state ofthe globin, and color (adapted from Fennema, 1985) Formulation guide to obtain 10% added water, 1% salt, and 0.5% sodium tripolyphosphate for 1.36-kg, and 2.27-kg meat samples Formulation guide to obtain a desired level of nitrite (1-20-ppm based on the meat weight) through dilution of nitrite stock solution (1000- ppm) with 10% water and 1.36-kg or 2.27-kg meat samples Panel color score, nitrosylhemochrome level, total pigment level, and Hunter color values of cooked beef round rolls formulated with 0-ppm and 9-12-ppm sodium nitrite Panel color score, nitrosylhemochrome level, total pigment level, and Hunter color values of cooked beef round rolls formulated with 0-ppm and ppm sodium nitrite Panel color score, nitrosylhemochrome level, total pigment level, and Hunter color values of cooked pork shoulder rolls formulated with 0- ppm and 4-7 -ppm sodium nitrite Panel color score, nitrosylhemochrome level, total pigment level, and Hunter color values of cooked turkey breast rolls formulated with 0- ppm and 1-4-ppm sodium nitrite Panel color score, nitrosylhemochrome level, total pigment level, and Hunter color values of cooked chicken breast rolls formulated with 0- ppm and 1-4-ppm sodium nitrite Summary of minimum parts per million added nitrite at which pink color was detected by trained panel and by Hunter color meter, and at which nitrosylhemochrome was extracted by acetone for beef, pork, turkey, and chicken samples

9 10. Minimum level of nitrite where pink color was detected by trained panelists, nitrosylhemochrome level in ppm at the identified level of nitrite, total pigment for samples and percent nitrosylation where pink color was detected by panelists Trained panelist color scores for beef samples containing 0- and ppm sodium nitrite (three replicate samples with fourteen trained panelists per replicate) One-way analysis of variance for trained panelist color score of beef samples containing 0- and 9-12-ppm sodium nitrite Nitrosohemochrome concentration of beef samples containing 0- and 9-12-ppm sodium nitrite (three replicate samples with duplicate testing) One-way analysis of variance for nitrosohemochrome concentration of beef samples containing 0- and ppm sodium nitrite Total pigment concentration of beef samples containing 0- and ppm sodium nitrite (three replicate samples with duplicate testing) One-way analysis of variance of total pigment concentration for beef samples containing 0- and 9-12-ppm Hunter color meter "L" values (lightness) for beef samples containing 0- and 9-12-ppm sodium nitrite (three replicate samples with three measurements per replicate) One-way analysis of variance of Hunter color "L" (lightness) values for beef samples containing 0- and 9-12-ppm Hunter color meter "a" values (redness) for beef samples containing 0- and 9-12-ppm sodium nitrite (three replicate samples with three measurements per replicate) One-way analysis of variance of Hunter color "a" (redness) values for beef samples containing 0- and 9-12-ppm Hunter color meter "b" values (yellowness) for beef samples containing 0- and 9-12-ppm sodium nitrite (three replicate samples with three measurements per replicate) One-way analysis ofvariance of Hunter color "b" (yellowness) values for beef samples containing 0- and 9-12-ppm Vlll

10 23. Trained panelist pink color scores for beef samples containing 0- and ppm sodium nitrite (three replicate samples with twelve trained panelists per replicate) Summary of one-way analysis of variance for beef samples (0- and ppm) trained panelist scores Nitrosohemochrome concentration of beef samples containing 0- and ppm sodium nitrite (three replicate samples with duplicate testing) One-way analysis of variance for nitrosohemochrome levels of beef samples containing 0- and ppm sodium nitrite Total pigment concentration ofbeefsamples containing 0- and ppm sodium nitrite (three replicate samples with duplicate testing) One-way analysis of variance for total pigment levels of beef samples containing 0- and ppm sodium nitrite Hunter color meter "L" values (lightness) for beef samples containing 0- and ppm sodium nitrite (three replicate samples with three measurements per replicate) One-way analysis of variance of Hunter color "L" (lightness) values for beef samples containing 0- and ppm sodium nitrite Hunter color meter "a" values (redness) for beef samples containing 0- and ppm sodium nitrite (three replicate samples with three measurements per replicate) One-way analysis of variance of Hunter color "a" (redness) values for beef samples containing 0- and ppm sodium nitrite Hunter color meter "b" values (yellowness) for beef samples containing 0- and ppm sodium nitrite (three replicate samples with three measurements per replicate) One-way analysis of variance of Hunter color "b" (yellowness) values for beef samples containing 0- and ppm sodium nitrite Trained panelist color scores for pork samples containing 0- and 4-7- ppm sodium nitrite (three replicate samples with fourteen trained panelists per replicate) IX

11 36. One-way analysis of variance of trained panelist color scores for pork samples containing 0- and 4-7-ppm sodium nitrite Nitrosohemochrome concentration for pork samples containing 0- and 4-7-ppm sodium nitrite (three replicate samples with duplicate testing) One-way analysis of variance ofnitrosohemochrome concentration for pork samples containing 0- and 4-7 -ppm Total pigment concentration for pork samples containing 0- and 4-7- ppm sodium nitrite (three replicate samples with duplicate testing) One-way analysis of variance of total pigment concentration for pork samples containing 0- and 4-7-ppm sodium nitrite Hunter color meter "L" values (lightness) for pork samples containing 0- and 4-7-ppm sodium nitrite (three replicate samples with three measurements per replicate) One-way analysis of variance of Hunter color "L" (lightness) values for pork samples containing 0- and 4-7-ppm sodium nitrite Hunter color meter "a" values (redness) for pork samples containing 0- and 4-7 -ppm sodium nitrite (three replicate samples with three measurements per replicate) One-way analysis of variance of Hunter color "a" (redness) values for pork samples containing 0- and 4-7-ppm sodium nitrite Hunter color meter "b" values (yellowness) for pork samples containing 0- and 4-7 -ppm sodium nitrite (three replicate samples with three measurements per replicate) One-way analysis of variance of Hunter color "b" (yellowness) values for pork samples containing 0- and 4-7-ppm sodium nitrite Trained panelist color scores for turkey samples containing 0- and 1-4- ppm sodium nitrite (three replicate samples with twelve trained panelists per replicate) One-way analysis of variance of trained panelist color scores for turkey samples containing 0- and 1-4-ppm sodium nitrite X

12 49. Nitrosohemochrome concentration for turkey samples containing 0- and 1-4-ppm sodium nitrite (three replicate samples with duplicate testing) One-way analysis of variance ofnitrosohemochrome concentration for turkey samples containing 0- and 1-4-ppm sodium nitrite Total pigment concentration for turkey samples containing 0- and 1-4- ppm sodium nitrite (three replicate samples with duplicate testing) One-way analysis of variance of total pigment concentration for turkey samples containing 0- and 1-4-ppm sodium nitrite Hunter color meter "L" values (lightness) for turkey samples containing 0- and 1-4-ppm sodium nitrite (three replicate samples with three measurements per replicate) One-way analysis ofvariance of Hunter color "L" (lightness) values for turkey samples containing 0- and 1-4-ppm sodium nitrite Hunter color meter "a" values (redness) for turkey samples containing 0- and 1-4-ppm sodium nitrite (three replicate samples with three measurements per replicate) One-way analysis of variance of Hunter color "a" (redness) values for turkey samples containing 0- and 1-4-ppm sodium nitrite Hunter color meter "b" values (yellowness) for turkey samples containing 0- and 1-4-ppm sodium nitrite (three replicate samples with three measurements per replicate) One-way analysis of variance of Hunter color "b" (yellowness) values for turkey samples containing 0- and 1-4-ppm sodium nitrite Trained panelist pink color scores for chicken samples containing 0- and 1-4-ppm sodium nitrite (three replicate samples with ten panelists for one replicate and nine panelists for two replicates) One-way analysis of variance of trained panelist color scores for chicken samples containing 0- and 1-4-ppm sodium nitrite Nitrosohemochrome concentration for chicken samples containing 0- and 1-4-ppm sodium nitrite (three replicate samples with duplicate testing) Xl

13 62. One-way analysis of variance of nitrosohemochrome pigment concentration for chicken samples containing 0- and 1-4-ppm sodium nitrite Total pigment concentration for chicken samples containing 0- and 1-4- ppm sodium nitrite (three replicate samples with duplicate testing) One-way analysis of variance oftotal pigment concentration for chicken samples containing 0- and 1-4-ppm sodium nitrite Hunter color meter "L" values (lightness) for chicken samples containing 0- and 1-4-ppm sodium nitrite (three replicate samples with three measurements per replicate) One-way analysis of variance of Hunter color "L" (lightness) values for chicken samples containing 0- and 1-4-ppm sodium nitrite Hunter color meter "a" values (redness) for chicken samples containing 0- and 1-4-ppm sodium nitrite (three replicate samples with three measurements per replicate) One-way analysis of variance of Hunter color "a" (redness) values for chicken samples containing 0- and 1-4-ppm sodium nitrite Hunter color meter "b" values (yellowness) for chicken samples containing 0- and 1-4-ppm sodium nitrite (three replicate samples with three measurements per replicate) One-way analysis of variance of Hunter color "b" (yellowness) values for chicken samples containing 0- and 1-4-ppm sodium nitrite Xll

14 Xlll LIST OF FIGURES Figure Page 1. Regression of total pigment vs minimum nitrite level causing pinking in cooked meats

15 INTRODUCTION Salt curing was one of the first preservation methods for meat. Accidental contamination of salt by saltpeter (potassium nitrate) increased the storage life and changed the flavor of the preserved meat. Today, nitrite and/or nitrate are added to meat products to obtain desirable cured meat color and flavor. Yet, in uncured meat processing situations, the pink cured meat color is undesirable. To some consumers, pink color represents an undercooked and/or unsafe product. Centuries ago, contamination of meat with nitrate/nitrite may have been desirable, but in a modem, consumer-driven marketplace, pinking due to nitrite contamination can result in economic losses to meat processors. Pink color of cooked meat can be due to many different things: nitrosylhemochrome resulting from nitrite contamination of ingredients, undenatured myoglobin, and denatured globin hemochromes (Cornforth, 1991). In order to prevent unwanted pink color, the cause must first be determined. Much research has been done to determine the causes, and corrections in processing methods have been made. Trout (1989) showed that salt and sodium tripolyphosphate increased the percent denatured myoglobin, which reduced pinking from undenatured myoglobin. In contrast, a high ph decreased the percent denatured myoglobin and increased pinking. Nitrite contamination is a relatively common cause of pinking problems. Well water, direct dried soybean proteins, packaging materials, whey powder, and other ingredients or spices can all contain nitrate or nitrite (Pool, 1956; Brant, 1984; Scriven et al., 1987). Recently, nitrogen dioxide (gas) has been shown to cause surface pinking

16 2 on beef or turkey cooked in a gas oven (Cornforth et al., 1998). As little as 2-ppm nitrite develops a pink color in cooked turkey breast (Ahn and Maurer, 1989). Direct dried soy proteins, which are exposed to nitrous oxides during drying, have high levels of nitrate and nitrite (311 and 47 -ppm, respectively; Cornforth, 1996). It is common practice to add soy protein to processed meats at rates of approximately 2%. Based on the above data, if 2% soy protein were added to a processed meat, it would contain about 6-ppm nitrate and 1-ppm nitrite. The question then raised is, "Is this enough to cause a significant pinking problem in cooked turkey products?" In pork or beef products about 25-ppm nitrite is needed for development of a stable cured pink color, and at least 100-ppm is needed for development of cured meat flavor (MacDougall et al., 1975). Fox (1987) reported that as little as 4-6-ppm nitrite caused curing, but at least ten times that amount is required to produce the cured meat (pink) color and prevent fading. However, documentation of the amount of nitrite (ppm) required to develop a pink color in the major animal meat species has not been accomplished. This information is needed so that processors of cooked meats know the nitrite levels of concern. The results of this research will be used by the meat industry to determine how much nitrite, if any, can be tolerated in uncured cooked meat products.

17 3 OBJECTIVES The objective of this study was to determine the minimum amount of nitrite (in ppm) that produces a visually detectable pink color in processed meat rolls made from beef round, pork shoulder, turkey breast, and chicken breast as detected by a trained panel. Instrumentally detectable changes in color and extractable nitrosylhemochrome pigment were followed in all samples to confirm visually detectable pinking. Nitrosylhemochrome pigment extraction confirmed that visually detected pink color was due to nitrosylhemochrome rather than some other cause. Total pigment was also determined for all samples because pigment content may affect the visual detection of pink color and the extraction of nitrosylhemochrome.

18 4 LITERATURE REVIEW Hemoglobin and myoglobin Throughout the process of converting live muscle to a consumable product, meat color is very important. Color can affect the quality grade and value of the carcass in the slaughter plant. In the grocery stores, unacceptable color means reduced prices or costs of additional processing. On the table, color can be the difference between meat products being tasted or wasted. To consumers, color is a quality standard associated to freshness, wholesomeness, and healthiness. There are two main pigments in meat: hemoglobin, the pigment of the blood, and myoglobin, the pigment of the muscle. Myoglobin:hemoglobin ratio can vary greatly in meat depending on the degree of freshness. Myoglobin is linked intercellularly with muscle tissue, but hemoglobin is linked with the fluid and can easily become lost in the blood or exudate. It has been observed in beef directly after slaughter that the myoglobin constitutes 58.4% of the total pigment (Livingston and Brown, 1981 ). In contrast, myoglobin constitutes 80-90% of the total pigment in well bled muscle tissue (Hedrick et al., 1994). Since the hemoglobin is lost rather easily during handling and storage, the color of a meat sample can fade if the initial myoglobin:hemoglobin ratio is low. However, assuming that most of the hemoglobin is lost initially, most color changes in meat are due to reactions of myoglobin with other muscle components (Livingston and Brown, 1981). Other pigments are also present in meat, but their contribution to color is minimal (Fennema, 1985).

19 5 The structure of myoglobin consists of a globular protein portion (globin) and a nonprotein portion (heme) containing an iron center and a planar porphyrin ring. One hemoglobin is essentially four myoglobins linked together, yet hemoglobin has a positive cooperativity of binding oxygen. This cooperativity binding of oxygen arises from the ligand binding state of one heme affecting the ligand binding state of adjacent heme groups in the hemoglobin molecule, which results from conformational changes of the hemoglobin protein after binding the initial oxygen (V oet and Voet, 1990). Meat color chemistry Bacterial growth, 0 2 and C0 2 levels, packaging methods, ph, light, and temperature have an influence on raw and cooked meat products. These factors do not change or directly affect meat color. Instead, these factors affect the "iron center" of the heme and the "iron center" chemistry determines the color. The oxidation state and type of ligand bound to the iron center determine the color and reactivity of myoglobin (Livingston and Brown, 1981). The protein myoglobin contains one heme moiety--one iron atom per molecule. Iron is a third-row transition metal, meaning that it has unfilled energy levels below its valence electron levels. The transition of electrons from filled to "3d" orbitals accounts for the visible absorption of light, and thus the color of myoglobin complexes (Livingston and Brown, 1981). The iron atom with its eight valence electrons (because of its electronegativity) may lose two valence electrons to form ferrous (Fe 2 +) iron or three electrons to form ferric (Fe 3 +) iron (Livingston and Brown, 1981). Ferric iron has a stronger pull on its electrons; therefore, it causes a shorter resonant electron wavelength. The wavelength of the iron's electrons will

20 6 match with the wavelength of visible light, absorbing some and reflecting the color that we see (Livingston and Brown, 1981 ). Table 1 shows the many combinations of meat color that can be achieved at different oxidation states and porphyrin ring conditions (Fennema, 1985). Table!--Pigments found in fresh, cured and cooked meats, mode of formation, state of iron, state of the globin, and color (adapted from Fennema, 1985) State of State of Pigment Mode of formation the iron the globin Color Myoglobin Reduction of metmyoglobin; Fe 2 + Native Purplish deoxygenation of red oxymyoglobin Oxymyoglobin Oxygenation of myoglobin Fe 2 + Native Bright red (bloom) Metmyoglobin Oxidation of myoglobin, Fe 3 + Native Brown oxymyoglobin Nitric oxide Combination of myoglobin Fe 2 + Native Bright myoglobin with nitric oxide red (pink) Nitrosyl- Combination of Fe 3 + Denatured Brown Metmyoglobin metmyoglobin with excess nitrite Globin Effect of heat, denaturing Fe 3 + Denatured Brown hemichromogen agent on myoglobin, oxymyoglobin, metmyoglobin and hemochromogen Nitric oxide Effect of heat, salt on nitric Fe 2 + Denatured Bright hemochromogen oxide myoglobin red (pink)

21 7 The three most common fresh meat pigments are myoglobin (purple red), oxymyoglobin (bright red), and metmyoglobin (greyish or brown). Myoglobin (Fe 2 +) radiates a purple or dark red color. Upon exposure to the atmosphere, oxygen combines with myoglobin to form oxymyoglobin (Fe 2 +). Oxymyoglobin, commonly called "bloom," is the fresh meat pigment that most consumers prefer in fresh meat. Eventually oxidation of the iron to its ferric form (Fe 3 +) occurs, converting the bright cherry red pigment into the brownish metmyoglobin (Fe 3 +; Fennema, 1985; Pearson and Gillet, 1996). Cured meat color The cured meat pigment is formed from myoglobin by two processes: first, the addition of nitrite or nitrate, and second, the thermal denaturation ofthe globin (Pearson and Gillet, 1996). When nitrite is added to meat, it combines with water to form nitrous acid (HN0 2 ). Nitrous acid then combines with myoglobin. Immediately, metmyoglobin begins to form due to the oxidation of the iron center, and nitric oxide (NO) and water (H 2 0) are formed. The nitric oxide combines with metmyoglobin to form nitrosylmetmyoglobin. It is well known that the cured meat (pink) color is due to the sixth position ligand being occupied by nitric oxide (NO) (Killday et al., 1988). Nitrosylmetmyoglobin through the process of internal reduction can be converted into nitrosylmyoglobin, which is the bright pink color of uncooked cured meat. Nitric oxide binds to the heme iron via the nitrogen atom to form a stable bond with the heme iron. Heat denaturation of either nitrosylmetmyoglobin or nitrosylmyoglobin will cause the common pink color of cooked cured meats. It is well known that the cured meat (pink)

22 8 color is due to the sixth position ligand being occupied by nitric oxide (NO) (Killday et al., 1988). Nitric oxide can combine with both ferric and ferrous iron on unhindered hemoproteins and hemilhemochromatogens, and under special situations with free heme/hematin (Giddings, 1977). Antonini and Brunori ( 1971) showed that the affinity ofno for Mb and Hb is exceptionally high, over 100 times greater than CO, which in turn has a much greater affinity than 0 2 Yet over time, 0 2 replaces the NO and oxidizes the iron, causing brown color. In the absence of 0 2, the NO complexes are very stable. Even though they are photodissociable, the dissociation rate is so low that it is usually insignificant. However, in the presence of 0 2, there is significant cured meat fading due to oxidation or photo-catalyzed dissociation ofno (Tarladgis, 1962). Fading was thought to be caused by withdrawal of electron density from iron to porphyrin, thus weakening the Fe-NO bond and leaving the iron susceptible to oxidation. Tarladgis (1962) also theorized through optical and EPR spectroscopy that the cured meat pigment was attributed to a Di-NO complex with heme iron. More recent studies done by Killday et al. (1988) showed through mass spectroscopy and other studies that there could only be a single NO attached to the heme iron. Cured meat attributes For decades nitrites/nitrates have been used and studied extensively in the meat industry. Accidental contamination of nitrate in the form of saltpeter led to the discovery of the usefulness of nitrite/nitrate in obtaining desirable cured meat products. Lehmann (1899) was the first to show that nitrites were also capable of inducing cured meat color. Hoagland (1908) correctly concluded that microbial reduction of nitrate to

23 9 nitrite was necessary for cured meat color development. This was conclusively shown by Walters and Taylor (1964), who showed that sterile muscle tissue cannot convert nitrate to nitrite, but in the presence of bacteria, nitrate is reduced to nitrite and nitric oxide. Fox ( 1966) studied the use of nitrite/nitrates as curing agents. Nitrites/nitrates retard microbial growth, induce the cured meat flavor, and cause the well accepted cured meat color. The effects of nitrites at levels of 0, 50, 100, and 200-ppm have been studied using ham (Froehlich et al., 1983). Froehlich et al. found that cured meat color (pink color) increased in proportion to added nitrite levels, but untrained panelists found cured color to be acceptable at 50-ppm added nitrite. Cured meat flavor intensity also increased with increased levels of nitrite. The contamination of meat, equipment, water supply, or ingredients by nitrates/nitrites has been suggested as causing the undesirable pinking of cooked meats (Brant, 1984; Everson, 1984; Scriven et al., 1987). Nitric oxide in the atmosphere of an electric oven has been shown to produce pink color (Pool, 1956). Cornforth et al. (1998) completed studies which showed that atmospheric nitric oxide is not the cause of pink ring defect in roast turkey and beef, but that nitrogen dioxide is the agent that causes pink ring defect. In 1925, the United States Department of Agriculture (USDA), based on comparison studies with nitrate, established the maximum level of nitrite in cured meat products at 200-ppm. In the 1970's, due to health concerns about nitrosamine formation in cured meats cooked at high temperatures, maximum levels were reduced to 120-ppm in bacon and 156-ppm in other cured meats (USDA, 1975, 1978a, b). Further investigative studies showed that vegetables are much higher in nitrate than are

24 10 meat products (Cassens, 1995). Gangolli et al. (1994) showed vegetables constitute a major source of nitrate, providing more than 85% ofthe average daily human dietary intake. There have been efforts in the meat industry to reduce the residual levels of nitrite in cured meats due to the possibility of carcinogen formation (nitrosamine) when the meat is cooked at high temperatures. Cassens (1995) concluded that there is about 1/10 the residual nitrite in modem cured meat products compared to the 1970's. Residual levels are found to be much lower than previously thought. Yet, unsuccessfully, research continues to try and find a substitute for nitrites/nitrates in cured meat products.

25 11 MATERIALS AND METHODS Experiment design The experiment included the four major processed animal meat species, including beef, pork, turkey, and chicken. These four meat species vary greatly in pigment to the extremes of highly pigmented (beef) to almost no pigment (chicken). For each meat type, we completed preliminary studies to narrow the range in which the meat turned pink. After the range was narrowed, our studies established the minimum levels of nitrite at which pink color could be detected by a trained panel and a colorimeter. We then conducted a final study to detect with acetone extraction the minimum level of nitrite which caused nitrosylhemochrome. The final studies involved an analysis oftriplicate meat rolls prepared from beefwith 0, 9, 10, 11, 12, 13, 14, 15, and 16-ppm added nitrite, from pork with 0, 4, 5, 6, and 7-ppm added nitrite, and from turkey and chicken breast with 0, 1, 2, 3, and 4-ppm added nitrite. Sample preparations Meat sample preparation. Raw meats were purchased from local distributing companies. If the meat were frozen, it was tempered for at least 24 h in a 2 o C (3 5 o F) cold room before further processing. Meat samples were chopped into approximately 5-cm cubes and ground twice through a grinder with a 25-mm plate (Model 4152, Hobart Mfg. Co, Troy, OH). The meat was then measured into 2.27-kg samples. Non-meat ingredients were added as percent meat weight as follows: 10% water, 1% salt, and 0.5% sodium tripolyphosphate. Only distilled water was used in

26 12 preparation of the samples. Food grade sodium tripolyphosphate and nitrite free salt were used. The non-meat ingredient levels (10% water, 1% salt, and 0.5% phosphate as a percent meat weight) were typical of many uncured processed meat products. Table 2 shows the amount of water, salt, and sodium tripolyphosphate added to kg and 2.27-kg samples. Preparation of non-meat ingredients included the following steps: (1) In a 600-ml glass beaker, the appropriate amount of distilled water (Table 3) was heated to greater than 52 oc (125 F). (2) Sodium tripolyphosphate in the appropriate amount was added and stirred until fully dissolved (Table 2). (3) Salt was then added in the appropriate amount (Table 2). (4) The water, salt, and phosphate solution was allowed to cool down before adding the required amount of nitrite stock solution (Table 3). (5) After mixing until completely dissolved, the non-meat ingredients were immediately added to the comminuted meat samples. Added ingredients and meat samples were mixed thoroughly by hand massaging and stirring for approximately 2 min until the meat had absorbed all added ingredients. Meat samples were stuffed into 15-cm clear plastic casings and stored in a 2 C (35 F) cooler 8-15 h until cooking could be completed. In order to be consistent, all samples Table 2--Formulation guide to obtain 10% added water, 1% salt and 0.5% sodium tripolyphosphate for 1.36-kg and 2.27-kg meat samples. Ingredient Water Salt Sodium tripoly phosphate 1.36 kg meat sample (g) kg meat sample (g)

27 Table 3--Formulation guide to obtain a desired level of nitrite (1-20 ppm based on the meat weight) through dilution of nitrite stock solution (1000 ppm) with 10% added water and 1.36-kg or 2.27-kg meat samples kg meat sam2le 2.27 kg meat sam2le Level ofnitrite ml ofnan0 2 ml ofnan0 2 ml ofwater { PPM2 stock solution ml ofwater stock solution were placed on racks and cooked in a 200-kg capacity smokehouse (V ortron, Inc., Beloit, WI) to an internal temperature of 74 oc (165 F), typical of commercial turkey rolls (Dobson and Cornforth, 1992). After cooking, meat samples were stored in a 2 oc (35 F) cooler until analysis was completed. Meat samples were never stored longer than 3 days before analysis was completed. Analysis was completed in the following order: trained panelist evaluations, Hunter color measurements, and nitrosylhemochrome acetone extraction.

28 14 Nitrite stock solution. Nitrite stock solution was prepared by dissolving 1 gram of sodium nitrite into 1 liter distilled water. Thus, the stock solution concentration was 1,000-ppm nitrite. In order to dilute it to desired test levels, a table was developed and followed throughout the experiment (Table 3). The desired nitrite level (ppm) can be found on the table and corresponding values for the amount of stock solution (1,000-ppm nitrite) and water can be determined. As the amount of nitrite stock solution increased, the amount of added water needed to maintain 10% added water in the samples decreased. Table 3 shows the amount of water and nitrite stock solution for nitrite levels between 1-ppm and 20-ppm for both 1.36-kg and 2.27-kg samples. Analysis of samples Trained panel ratings of pink color intensity. At least 10 panelists with normal color vision (self-evaluated) were selected from the students, faculty, and staff at Utah State University. Panelists received training prior to analyzing samples. All panelists received a score sheet prior to evaluating samples. Four different samples of cured and uncured meats (ham and pastrami, cured; roast turkey and beef, uncured) were purchased from the Utah State University Deli Bar. Panelists compared pink color intensity of the cured and uncured meat samples and rated control samples using the following scale: 1 = no pink, 2 = slightly pink, 3 = moderately pink, 4 = very pink, and 5 = extremely pink. Training also focused on the rating of the control samples. Panelists were instructed that uncured meats (roast beef or turkey) should be given a 1 =no pink rating. Ham-like pinkness should be given a 3 =moderately pink or 4 =

29 15 very pink rating. Cured beef pastrami was the most pink control with a rating of 5 = extremely pink. At the end of each training session, panelists were given the opportunity to ask questions and discuss the ratings of the control samples. After completion of training, experimental samples were cut into slices 1 em thick and placed on 15-cm diameter styrofoam plates, labeled with a three-digit sample number. Panelists immediately rated the samples on the previously described color scale. Upon completion ofthe evaluation, panelists were given ice cream coupons redeemable at the USU Dairy Bar. Hunter color meter value. A Hunter Lab Digital Color Difference Meter (model D25D2) (Hunter Associates Laboratory, Reston, VA) was standardized using a pink standard plate ( "L" = 66.8, "a"= 21.4, and "b" = 12.0). The Hunter color meter was used to obtain measurements of "L" (lightness), "a" (redness), and "b" (yellowness). One meat sample per treatment was sliced to approximately 1 em thick and immediately placed into the machine to minimize fading of the light-sensitive pink color. After recording L, a, and b scores, the sample was rotated 90 degrees twice and L, a, and b readings were taken after each rotation on the same site. Pigment extraction. Mononitrosylhemochrome (cured meat pigment) and total pigment was extracted in acetone using the method developed by Homsey (1956) and modified by Kramlich et al. (1973). Reagents used in this experiment were prepared as follows: 1. Acetone A: Place 90 ml distilled water in a 1 L volumetric flask. Bring to volume with acetone (96%) then mix.

30 16 2. Acetone B: Place approximately 50 ml distilled water in a 100 ml volumetric flask. Mix 20 ml concentrated HCl with the water and bring to volume with water. Transfer the diluted HCl to a 1 L volumetric flask, add acetone, mix, and bring to volume with acetone (96%). Nitrosylhemochrome pigment determination. Testing of meat samples were done in subdued light to lessen pigment fading. After removing oxidized surfaces from the meat samples, approximately 20 grams was diced into 3-mrn cubes. For each treatment, duplicate samples of2.0 grams were weighed and placed in a 25-ml glass test tube containing 9 ml acetone A solution and capped to prevent evaporation. The sample was macerated for 1-2 min using a 20-cm glass stirring rod. The screw-on cap was placed on the test tube. After waiting 10 min, the sample was filtered through a Whatman 42 filter paper into a test tube. After transferring into a 1-ml cuvette, the absorbance was read at 540 nm using a spectrophotometer (Spectronic 21 UV-VIS, Milton Roy, Rochester, NY). The nitrosohemochrome pigment was calculated using the following equation: PPM nitrosohemochrome pigment = Abs 540 X 290 Total pigment determination. After removing oxidized surfaces from the meat samples, approximately 20 grams was diced into 3-mm cubes. For each treatment, duplicate samples of 2.0 grams were weighed and placed in a 25-ml glass test tube containing 9 ml acetone B solution and capped to prevent evaporation. The sample was macerated for 1-2 min using a 20-cm glass stirring rod. The screw-on cap was placed firmly on the test tube to prevent evaporation of acetone. After waiting at

31 17 least 1 h, the sample was filtered through a Whatman 42 filter paper into a test tube and transferred into a 1-ml cuvette. The absorbance was read at 640 nm using a spectrophotometer (Spectronic 21 UV-VIS, Milton Roy, Rochester, NY). The total pigment was calculated using the following equation: PPM total pigment = Abs 640 X 680 Statistical analysis. After completion of the lab work, one-way analysis of variance (ANOV A) was completed using Quatro Pro (Novel Inc. 1994). To analyze for significant difference among the means, the least significant difference was calculated (Neter and Wasserman, 1974).

32 18 RESULTS AND DISCUSSION The mean values and statistical comparisons for beef, pork, turkey, and chicken rolls are presented in Tables 4-8, respectively. The minimum levels of nitrite at which visually and instrumentally detectable pink color and extractable pigment are identified are presented in Table 9. Beef round The panel clearly distinguished a pink color difference at 14-ppm added nitrite as compared to the 0-ppm controls. Hunter color meter "L" (lightness) values were not different from controls until samples contained 13-ppm nitrite (Table 5). Hunter Table 4--Panel color score, nitrosylhemochrome level, total pigment level, and Hunter color values of cooked beef round rolls formulated with 0-ppm and 9-12-ppm sodium nitrite NO Hemechrome Hunter color mete~ Nitrite Trained pigment (ppm) panel score 1 (ppm) "L" "a" "b" 0 1.3± A A± ± A± ± A± AC± ± A± ± A± ± ± ± ± A± c± ± A± ± ± AC± ± A± 0.5 LSDoos Trained panel scores: 1 = not pink, 2 = slightly pink, 3 =moderately pink, 4 = very pink and 5 = extremely pink. 2 Hunter color: "L" = lightness, "a" = redness and "b" = yellowness. A-cvalues in columns with the same superscript are not different (p<0.05).

33 Table 5 -Panel color score, nitrosylhemochrome level, total pigment level, and Hunter color values of cooked beef round rolls formulated with 0-ppm and ppm sodium nitrite NO Hemechrome Hunter color mete~ Nitrite Trained pigment (ppm) panel score 1 (ppm) "L" "a" "b" 0 1.4A± A± A± A± A± A± B±l B± A± A± B± c± A± B± B± AB± c 0 ± B± BC± c± B± ± c± ± BC± 0.3 LSDoos Trained panel scores: 1 =not pink, 2 =slightly pink, 3 =moderately pink, 4 =very pink and 5 = extremely pink. 2 Hunter color: "L" = lightness, "a" =redness and "b" = yellowness. A- 0 Values in columns with the same superscript are not different (p<0.05). 19 color "a" and "b" values were different from controls at 14-ppm nitrite. Acetone extraction of nitrosohemochrome showed a difference in pigment levels between controls and samples with 12-ppm or higher nitrite (Table 4). Thus, for beef samples, acetone extraction of cured meat pigment detected pigment before instrumental measurements or human subjects detected pink color. Pork shoulder Trained panelists rated the control and 4-ppm pork samples as different, with mean scores of 1.6 and 1.8, respectively (Table 6). Hunter color "L" values for samples with all levels of nitrite were not different from the control. Hunter color "a"

34 Table 6--Panel color score, nitrosylhemochrome level, total pigment level, and Hunter color values of cooked pork shoulder rolls formulated with 0-ppm and 4-7-ppm sodium nitrite NO Hemechrome Hunter color metez2 Nitrite Trained pigment (ppm) panel score 1 (ppm) "L" "a" "b" 0 1.6A ± A ± ± A± A± ± ± ± C± ± c ± ± ± AB ± ± ± c ± ± CD± C± E ± ± ± ± c± 0.3 LSDoos Trained panel scores: 1 =not pink, 2 =slightly pink, 3 =moderately pink, 4 =very pink and 5 = extremely pink. 2 Hunter color: "L" = lightness, "a" = redness and "b" =yellowness. A-Evalues in columns with the same superscript are not different (p<0.05). 20 values showed 4-ppm, 6-ppm, and 7-ppm samples different from the control. The 5- ppm samples were not different from the controls. At 6 ppm and above, the samples were consistently different from the control. Hunter color "b" (yellowness) values were lower than the control at nitrite levels, 4 ppm, 5 ppm, 6 ppm, and 7 ppm. Acetone extraction detected pink pigment at 4-ppm added nitrite. Nitrosylhemochrome extraction detected pigment in 4-ppm added nitrite samples, which was in agreement with the panel (Table 9). Hunter color measurements detected pinking at 6-ppm. Thus, for pork samples, instrumental measurements of pink color were not as sensitive as the panel detecting pink color or acetone extraction detecting pink pigment.

35 Table 7--Panel color score, nitrosylhemochrome level, total pigment level, and Hunter color values of cooked turkey breast rolls formulated with 0-ppm and 1-4-ppm sodium nitrite NO Hemechrome Hunter color metez2 Nitrite Trained pigment (ppm) panel score 1 (ppm) "L" "a" "b" 0 1.0A± A± A± A± A± A± A± AB± AB± B± B± A± B± BC± c± c± B± B± c± ± ± c± c± ± E± 0.9 LSDoos Trained panel scores: 1 =not pink, 2 =slightly pink, 3 =moderately pink, 4 =very pink and 5 = extremely pink. 2 Hunter color: "L" = lightness, "a" = redness and "b" = yellowness. A- Evalues in columns with the same superscript are not different (p<0.05). 21 Turkey breast Trained panelists easily identified the turkey breast control (0-ppm) by rating it a " 1" (Table 7). The panel did not distinguish a difference between the control and 1- ppm with means of 1.0 and 1.1, respectively. At 2-ppm, the panel rated the samples as different than the control with a mean of Hunter color "L" (lightness) values decreased as the level of nitrite in the samples increased. Hunter color "a" values for 0- ppm and 1-ppm were not different from each other with means of 5.4 and 5.8, respectively. The 2-ppm sample had a higher pinkness value than the control, but not higher than the 1-ppm sample. The 4-ppm sample had higher values than the 0-ppm, 1- ppm, 2-ppm, and 3-ppm samples. Hunter color "b" (yellowness) values were different

36 Table 8--Panel color score, nitrosylhemochrome level, total pigment level, and Hunter color values of cooked chicken breast rolls formulated with 0-ppm and 1-4-ppm sodium nitrite NO Hemechrome Hunter color metey-2 Nitrite Trained pigment (ppm) panel score 1 (ppm) "L" "a" "b" 0 1.1A± A± A± A± A± ± A± ± ± ± c± ± ± c± c± ± ± c± ± ± c± ± C± ± ± 0.2 LSDoos Trained panel scores: 1 =not pink, 2 =slightly pink, 3 =moderately pink, 4 =very pink and 5 = extremely pink. 2 Hunter color values: "L" = lightness, "a" = redness and "b" = yellowness. A-ovalues in columns with the same superscript are not different (p<0.05). 22 Table 9. Summary of minimum parts per million added nitrite at which pink color was detected by trained panel and by Hunter color meter, and at which nitrosylhemochrome was extracted by acetone for beef, pork, turkey, and chicken samples. PPM added nitrite Specie Trained panel Acetone extraction Hunter color meter Beef Pork Turkey Chicken 2 1