Concise Reviews in Food Science

Pink Color Defect in Poultry White Meatas Affected by Endogenous ConditionsK. HOLOWNIA, M.S. CHINNAN, AND A.E. REYNOLDS

ABSTRACT: The pinking defect in cooked, uncured meat has been a problem in the poultry industry for nearly 40years. Through the years, analyses of data revealed various processing factors that seem to influence the specificbiochemical conditions (pH, redox potential, denaturation, reacting ligands) of the meat that are related to thechemical state of the pigments in cooked meat, their structure, and reactivity. This review addresses endogenousconditions that affect the pigments’ reactivity, and research studies conducted on in situ conditions resulting inpinking in cooked meat. Future studies could be devised for understanding mechanisms leading to developingprocesses for reduction/elimination of the pink defect in cooked white poultry meat.

Keywords: pinking, defect, poultry, cooked meat

Introduction

WHEN THE PINK DEFECT (PINKING, PINKNESS, OR PINK TINGE) OF

cooked poultry meat occurs, white meat exhibits areas that re-tain a pink color even after the meat has been cooked to an internaltemperature far above that required by the Food Safety and Inspec-tion Service agency of the U.S. Dept. of Agriculture. Although thepinking is a cosmetic problem, it can greatly affect the purchasingbehavior of consumers. Consumers may interpret the pink discolor-ation as an indication of undercooked product that is unsafe to eat.Therefore, this defect can result in serious economic losses to the re-tailers, processors, and producers of poultry products. Froning (1995)presented an extensive review of many factors affecting poultrymeat color, and Maga (1994) reviewed the primary factors influenc-ing pinking. Factors related to the pink defect include (1) variousclasses and types of pigments (Fox 1966; Tappel 1957; Ledward 1974;Livingston and Brown 1981; Izumi and others 1982; Cornforth andothers 1986; Girard and others 1989; Ghorpade and Cornforth 1993;Smith 1998), (2) preslaughter factors such as genetics, feed, haulingand handling, heat and cold stress, and gaseous environment (Fron-ing and Hartung 1967; Froning and others 1968a, 1969a, 1978; Babjiand others 1982; Ngoka and others 1982; Sackett and others 1986),(3) stunning techniques (Ngoka and Froning 1982; Froning 1995;Young and others 1996b; Craig and others 1999), (4) incidental ni-trate/nitrite contamination through diet, water supply, freezing andprocessing equipment, and processing ingredients (Froning andothers 1968b, 1969b; Mugler and others 1970; Nash and others 1985;Ahn and others 1987; Fleming and others 1991; Heaton and others2000), (5) current industry procedures including the use of nonmeatingredients and cooking methods (Pool 1956; Froning and others1968c; Helmke and Froning 1971; Janky and Froning 1973; Trout1989; Ahn and Maurer 1989a, 1990a,b; Claus and others 1994; Corn-forth and others 1998), and recently, (6) irradiation of precookedproducts (Nam and Ahn 2002a, b).

All of the mentioned factors seem to change the specific in situconditions of the meat, including such characteristics as pH, reduc-ing conditions, degree of denaturation, and reactivity of endoge-nous meat compounds. These in situ conditions affect the chemi-cal state, structure, and reactivity of the pigments. Meat can beconsidered a light-scattering matrix of cellular material, myofibrillarproteins, connective tissue, and light-absorbing pigments (Mac-

Dougall 1970; Swatland 1983). Eventually the physical character-istics of meat such as its light scattering and absorbing propertiesare affected by the in situ condition and thereby influencing thefinal color of meat products. There is an extensive amount of knowl-edge and literature about pigments’ chemistry and reactions. Thisreview is an attempt to deal only with endogenous conditions thataffect the pigments’ reactivity and to present the research studiesconducted through the years on in situ conditions that affect pink-ing in cooked white poultry meat.

Pigments’ denaturation and reactions with ligandsThe intensity of reflected light and hence the color and appear-

ance of the meat are affected by a complex interaction betweenlight scattering, selective absorbance of the pigments, and the sam-ple structure (MacDougall 1970; Swatland 1983; Renerre 1990).Light absorption and color are consequences of both the resonanceof the prosthetic heme and the reaction with binding molecules(MacDougall 1982). The chemistry of myoglobin and its reactionwith ligands has been reviewed in general by Antonini and Brunori(1971) and with particular emphasis on meat color by Giddings(1977), Livingston and Brown (1981), and MacDougall (1982). Allferrous and ferric forms of myoglobin have 6 ligands bound to theiron cation. The heme pyrolle nitrogen occupies 4 of the 6 coordina-tion positions. In the native myoglobin derivatives, the 5th positionis occupied by histidine. This leaves the 6th position open for sub-stitution with ligands that can affect meat color. The ability of aligand to coordinate with the iron in native myoglobin is limitedbecause of the small cleft in the protein structure; that is, sterichindrance exists for a large ligand (Livingston and Brown 1981).Consequently, only small molecules (for example O, NO, CO) canbind to the iron center in the native myoglobin. Denaturation, theconversion of myoglobin and metmyoglobin to ferro- and ferri-hemochromes respectively, is the process that involves the unfold-ing of the native myoglobin proteins. This process allows the expo-sure of the heme group to potential ligands allowing it to react withthe iron center. Potential ligands can be formed when heat unfoldsthe polypeptide chains of proteins, causing exposure of active sidechains of the amino acids (Brown and Tappel 1957; Ahn and Maurer1990a). After denaturation of myoglobin, the heme groups will com-bine with certain amino acids, denatured protein, and many other

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nitrogen-containing substances to produce hemochromes. Thedenatured globin hemochromes have been characterized by manyauthors as (1) pinking pigments of well-cooked meats refrigeratedanaerobically, (2) a mixed complex of ferrous heme iron with nitro-gen from nicotinamide or from amines of denatured proteins, (3)insoluble and not extractable, and (4) measured by highly charac-teristic spectral curves with reflectance minima at about 530, 555,and 415 nm (Pool 1956; Akoyunoglou and others 1963; Brown andTappel 1957; Dymicky and others 1975; Cornforth and others 1986;Ghorpade and Cornforth 1993). The most likely hemochromeshave as their nonheme constituent either denatured globin or nic-otinamide or perhaps both (Brown and Tappel 1957). The fact thatcertain proteins have more affinity for hematin than others may berelated to the spatial arrangement of the histidine residue in theprotein. Histidine also has the ability to form stabilizing porphyrin-protein interaction via salt-linkages, hydrogen, and hydrophobicbonds (Ledward 1974).

Dymicky and others (1975) studied color formation in cookedmodel systems. Formation of the color is related to the nature of thesubstituent and its position on the ring. The substituted carbonylpyridines produce a pink to reddish coloration, similar to that of thenitrite-containing control. Nicotinic acid and esters in this studyform tan, light pink complexes. Imidazole also forms a stable pinkpigment. The imidazole residues may be supplied by the histidinegroups of the bound protein (Ledward 1974), the epsilon aminegroup of lysine, or the terminal alpha amino group of denaturedprotein. Histidine, lysine, and arginine all yield pink complexeswith absorption peaks at ~410 and ~535 nm and shoulders at ~580and 620 nm that are spectrally similar to cooked-meat hemoprotein(Ledward 1974). The added salt solubilizes myofibrillar proteins,and the heat increases the number of exposed reactable sidechains, thereby providing more chances for heme-complex forma-tion in the meat (Ahn and Maurer 1989a). According to Ahn andMaurer (1990a) histidine, cysteine, and methionine, or their side-chains from solubilized proteins, and vitamin B6 derivatives, arevery important constituents in the pinking of cooked meat. The pHrange of 6.1 to 6.4 is critical for heme complex formation of myoglo-bin and hemoglobin with naturally present ligands such as histi-dine, cysteine, methionine, nicotinamide, and solubilized proteins(Ahn and Maurer 1990a). Pink color development associated witha protein such as globin, nicotinamides, or albumin is possible afterheating and is dependent upon protein denaturation after cook-ing (Cornforth and others 1986). Claus and others (1994) studiedthe effects of cooking temperatures, chilling rate, and storage timerelative to the formation of the pink, denatured globin-hemo-chromes complex. They concluded that in ground turkey breastmeat containing 2% nicotinamide, sensory pinkness and CIE a*values are higher with an increasing endpoint temperature (71 to80 °C) and with a slower chilling rate of the cooked product. Nicoti-namide may be superior in terms of hemochrome forming ability toany other substances tested because it readily reacts under reduc-ing conditions in either neutral or basic pH solution (Brown andTappel 1957). Formation of ferrohemochromes is greatly influencedby pH, ligand concentration, and ligand electronegativity. The low-er the electron-donor power of the ligands the more the ferrousstate becomes stable compared to the ferric state (Akoyunoglouand others 1963; Falk 1964). Increasing electronegativity apparentlyenhances reactivity, provided that steric factor does not prevail(Akoyunoglou and others 1963).

One of the nitrogenous groups reacting with heme to form hemo-chrome is nitric oxide, which is used to preserve myoglobin in theferrous state. Nitrates and nitrites are commonly used in curedproducts. Even in uncured meat when a small but sufficient

amount of nitrite is present in meat it will react and form pink ni-trosopigment. The residual pink color may be caused by nitratemicrobial conversion to nitrite to form a nitric oxide myoglobin.When a high amount of nitrate in meat and/or processing waterand a long storage period are combined, the probability of the de-velopment of the pink color in the oven roasted turkey breast isvery high (Nash and others 1985; Ahn and Maurer 1987). The addi-tion of only 1-ppm nitrite causes a significant color change in ovenroasted turkey breast (Ahn and Maurer 1989a). Processing ingredi-ents such as dried soy isolates may contain a sufficient amount ofnitrite to react with meat pigment (Heaton and others 2000). Inaddition, ammonia present in live poultry production and refriger-ation systems was also a subject of study concerning undesiredpinking in cooked meat. Shaw and others (1992) presented thatexposure of raw pork loins to 20 mL of concentrated ammoniumhydroxide for 30 min resulted in formation of pink color after cook-ing. The discoloration was attributed to pink appearing reduceddenatured hemochromes as indicated by reflectance spectra. How-ever, earlier research by Sackett and others (1986) indicated noeffect of birds’ exposure to ammonia (25 to 100 ppm) on either rawor cooked color of chicken breast.

Carbon monoxide binds tightly to myoglobin, forming ferrouscarboxymyoglobin with a visible spectrum similar to that of oxymyo-globin and nitric oxide myoglobin. Carboxymyoglobin provides astable red color in meat even after the denaturation of proteins hadoccurred (Livingston and Brown 1981). Significant pinking of beefand turkey, especially on the surface, is related to carbon monoxidein gas ovens (Pool 1956; Cornforth and others 1998). In large roastsor rolls exposed to NO or CO gas, a pink ring of .25-in thick maydevelop and be responsible for what is called surface pinking. How-ever, in most cases the pink defect appears in the product’s interiorwhereas the extent of surface pinking depends on the product’scharacteristics (an intact muscle versus ground product). Irradia-tion as a processing preservation technique may produce a suffi-cient amount of carboxymyoglobin to be responsible for the pinkcolor formation (Nam and Ahn 2002b). Nam and Ahn (2002a) andKim and others (2002) characterized the color compounds that canbe generated by irradiation of raw turkey breast meat. CO provid-ed the most reducing conditions in meat resulting in formation ofpink carboxymyoglobin. Interestingly, pink color formed in the rawturkey breast meat remains after cooking only upon vacuum stor-age of raw meat (Du and others 2002). This implies that aerobic dis-play of irradiated meat could actually eliminate potential pinkingdue to irradiation. Also, carboxymyoglobin was found in vacuum-packed precooked turkey breast suggesting that CO as a 6th ligandcoordinating to heme may be responsible for formation of reducedpink hemochromes. Nicotinamide and nitrite are popular amongresearchers to induce pink color in meat when studying the pinkingphenomenon in white poultry meat (Schwarz and others 1997; Sles-inski and others 2000a,b). Since the pink hemochromes are formedwhen certain ligands bind to the heme pigments, a novel approachwas presented to bind selected ligands to the heme ring that do notcause a pink color (Schwarz and others 1997, 1999). Schwarz andothers (1997) found the effective ligands (diethylenetriamine, eth-ylenedinitrilo-tetraacetic acid disodium salt, trans 1,2-diaminocy-clohexane-N,N,N’,N’ tetraacetic acid monohydrate, calcium re-duced nonfat dried milk) in the presence of sodium nitrite andnicotinamide, the pink color formation agents. Schwarz and others(1999) concluded that addition of 50 ppm of ethylenedinitrilo-tet-raacetic acid disodium salt (EDTA) or diethylenetriamine pentaace-tic acid (DTPA) to uncured turkey breast muscle was sufficient toreduce pinking after cooking. There is an opportunity for somedairy proteins or some components of dairy proteins to reduce or

Concise Reviews in Food Science

Pink color defect in poultry white meat . . .

eliminate the pink defect (Slesinski and others 2000a,b), but themechanism for the reduction is not yet established.

Meat pHPrevious research has shown that muscle pH and meat color are

highly correlated. Higher pH values are observed in dark breastmuscle whereas low pH values are observed in light colored breastmuscle (Fletcher 1999a). Since muscle pH is related to the biochem-ical state of the muscle at the time of slaughter and during rigormortis development, the muscle pH influences both the light reflec-tance properties of the meat as well as the chemical reactions of themyoglobin. Most of the reactions which interconvert ferrous andferric forms of myoglobin are reversible and set up a “dynamic” colorcycle in meats (Fox 1966).

The rate and the extent of pH decrease during rigor mortis areinfluenced by intrinsic factors such as species, breed, muscle andanimal variability, as well as extrinsic factors such as environmentaltemperature and degree of stress (Lawrie 1985). Sackett and others(1986) found that the adaptation of birds to stress caused by carbondioxide exposure affects utilization of glycogen reserves, eventual-ly causing increased pH (5.84 to 5.91) of the raw breast meat whencompared to the pH (5.73) of the control group. The redness of rawmeat is also significantly higher in all meat from birds stressedthrough exposure to carbon monoxide prior to slaughter (Sackett andothers 1986). Heat stress and cold stress evidently affect meat pHand meat quality in a different way (Wood and Richards 1975; Fron-ing and others 1978; Babji and others 1982). According to Wood andRichards (1975) heat stress of broilers increases the rate of postmor-tem glycolysis that may produce pale meat color with low pH as alsoindicated by Babji and others (1982). Different results presented byFroning and others (1978) indicated that heat-stressed turkey pro-duced darker and redder breast muscle. However, shorter time ofheat exposure in their study compared to other studies could reducethe effect of accelerated glycolysis. Antemortem cold stress of birdsextends the period of postmortem glycolysis (Wood and Richards1975). Muscles derived from cold-stressed birds showed pH about6.06, lower shear values and color similar to control group resultinginto meat with better quality characteristics (Babji and others 1982).Electrical stunning and postmortem stimulation have significanteffect on the pH of raw meat and cooked meat; however, those differ-ences seem to diminish after 24 h postmortem (Craig and others1999). Even then, the problem of elevated pH influences early deb-oned poultry meat. According to Fletcher (1999a,b), a strong relation-ship exists between chicken breast meat color and muscle pH withfillets from the darker groups having significantly higher pH valuesthan the lighter groups. The lightness of cooked breast meat colorsis more closely associated with raw breast meat pH than with cookedmeat pH (Fletcher and others 2000).

At neutral pH for myoglobin and hemoglobin, the affinity ofheme for the protein is very high. Under normal physiological condi-tions, the heme is quite well protected from damage by being buriedin the protein’s cleft (Livingston and Brown 1981). The absorptionspectrum of deoxygenated myoglobin or hemoglobin is invariantover the pH range in which the pigment is stable (Antonini andBrunori 1971). The absorption of metmyoglobin is greatly influencedby higher pH, and, since myoglobin oxidizes readily, the influence ofalkaline pH is important in spectrophotometric studies (Bowen1949). The pigment reactions that are triggered by muscle pH alsomay contribute to the occurrence of the meat pink color defect.

Low pH muscle has an open structure, which allows for greateroxygen penetration (Hunt and Kropf 1987). The open structure isrelated to the tightness (different from toughness) of muscle struc-ture. Sarcoplasmic proteins are confined to the sarcoplasm around

the myofibrils which themselves enclose a solution of salts andmetabolites (Greaser and others 1969). As the pH falls myofibrilsshrink and loss of fluid from myofibrillar proteins increases extra-cellular space. This results in pushing out the protein-free solutioninto the sarcoplasm (Bendall and Swatland 1988; Renerre 1990)which translates into dilution of the soluble sarcoplasmic proteinsresulting in increased light scattering. In meat with a high light-scattering ability, such as low pH meat, light does not penetrate farinto the meat before being scattered; hence there is relatively littleabsorption by myoglobin and so the meat appears pale and light incolor (MacDougall 1970). The instability of myoglobin at a pH lowerthan 6.0 is related to globin unfolding and to partial loss of helixformation (Janky and Froning 1973). The pH affects enzymaticactivity of the mitochondrial system, thereby altering the oxygenavailability for heme reactivity (Livingston and Brown 1981; Ren-erre 1990). Myoglobin is less heat sensitive near its isoelectric point7.0 than at lower pH values (Young and others 1996b; Ledward1974). Janky and Froning (1972, 1973) studied the effects of pHand heat denaturation of turkey myoglobin. They found that myo-globin is more susceptible to denaturation when pH was < 6.0.Trout (1989) used a model system to show that the sensitivity ofmyoglobin to heat denaturation increases as muscle pH decreases.Autooxidation is accelerated by low pH (Livingston and Brown 1981;Gutzke and Trout 2002). Autoxidation rate increases with any con-ditions that destabilize the linkage of the heme to the protein moi-ety and lead to greater exposure of the heme.

High pH meat has a high water-binding capacity and is associat-ed with greater translucence and less light scattering. This lowerscattering allows greater light penetration and absorption whichmakes the meat appear darker and redder (MacDougall 1982; Ben-dall and Swatland 1988). At high pH (~6.1), the iron of heme is pre-dominantly in the ferrous state, and low pH accelerates ferrous ironconversion to the ferric state (Ahn and Maurer 1990b). Controllingoxidation to metmyoglobin includes maintaining a high final pH(Livingston and Brown 1981) but, at the same time, allowing great-er reactivity of heme pigments. The neutral and alkaline pH has aprotective effect on the denaturation behavior of myoglobin. In-creasing the pH from 5.5 to 7.0 markedly decreases the percentageof myoglobin denatured at a given temperature, except at a tem-perature where the myoglobin is completely denatured (Trout1989). Young and others (1996a) suggested that cooking poultrymeat before completion of the rigor process can lead to sufficientpH change such that the temperature required for myoglobin de-naturation increases above that normally achieved. At tempera-tures lower than 76 °C and meat pH greater than 6.0, there is aninsufficient denaturation of myoglobin. This implies that there isan increase of the myoglobin in its redder form which causes pinkdefect similar to that of an undercooked product (Trout 1989; Youngand others 1996a). A study by Ahn and Maurer (1990b) showed thata pH > 6.4 was favorable for the heme complex reactions of myoglo-bin and hemoglobin with most of naturally present ligands (histi-dine, cysteine, methionine, nicotinamide, solubilized proteins). AtpH 7.4 and higher heme complex forming reactions are observed ina myoglobin solution with no additional ligands added (Ahn andMaurer 1990b). As the state of the pigment is largely decided bythe meat pH, the reaction of the heme with other reactants is alsoaffected. If the meat pH is higher than 6.3, the side chains of histi-dine, cysteine, and methionine from meat proteins, and nicotina-mide start to form heme complexes and exhibit a pink coloration ofmeat (Ahn and Maurer 1990b).

Salts and phosphates increase the functional properties of meatproteins, and hence the functional properties of the meat products,by changing the conformation of the constituent proteins (Hamm

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1960, 1971; Damodaran and Kinsella 1982). Because of the change inconformation the proteins do not aggregate extensively when heat-ed but form an ordered, 3-dimensional lattice structure (Siegel andSchmidt 1979). Salt increases the potential for myoglobin oxidationby decreasing the buffering capacity of meat, and by promoting low-er oxygen tensions in meat (Seideman and others 1984). However,the combination of NaCl and phosphate generally has a beneficialsynergistic effect on product color. Sodium chloride facilitates therelease of structural proteins from the muscle cells at the surface ofthe meat during mechanical treatment. Phosphates may also causeslowing of the rate of pH decline. Together, the effects seem to berelated to pH elevation, ionic strength, metal ion chelation, andstructural interactions with myofibrillar proteins (Trout and Schmidt1984; Li and others 2001). When treated with salt and sodium tripoly-phosphate a significant increase in muscle pH occurs. The effect ofcooking on pH however, depends on the aging time for the muscle(Young and others 1996a). According to Yang and Chen (1993), chick-en meat is darkened significantly by the addition of phosphate, andthe darkness was proportional to the amount of phosphate added.Alkaline phosphates in particular have sufficient buffering capaci-ty to counteract pH drop (Young and others 1999). Phosphates alsoreduce the rate of pigment and lipid oxidation by the chelation of freeiron, a lipid prooxidant (Pearson and others 1977). This in turn wouldreduce oxidation of myoglobin because myoglobin, is also oxidizedby lipid oxidation byproducts. Increasing the pH of fresh meat above6.0 greatly reduces the rate of lipid and myoglobin oxidation (Yasoskyand others 1984). A meat pH that is high enough to facilitate myo-globin reduction and decrease the susceptibility of both lipid andpigments to oxidation will consequently cause pink or reddish discol-oration in cooked meats.

Meat protein functionality is attributed to changes in the electro-static interactions between charged groups in the muscle protein(Hamm 1971). In addition, both autoxidation of myoglobin anddenaturation of proteins are affected by pH. The denaturation is aprerequisite for forming hemochromes, the pigments of cookedmeat. When myoglobin is denatured, several types of amino acidsside chains can coordinate to heme iron (Livingston and Brown1981). Most of the ligands that bind to myoglobin have an elec-tronegative atom (N or O) that donates electrons to the metal (Tar-ladgis 1962; Livingston and Brown 1981). Electronegativity of theamino acids side chains (dissociation of hydrogen ion from a sidechain) may be increased further by increased pH. Therefore, pH isone of the factors in heme complex formation with amino acids orprotein ligands. With increased pH, ligands may donate electronsto ferrous iron to form low spin sigma bonding that is characteristicof pink stable ferrohemochromes (Akoyunoglou and others 1963;Ahn and Maurer 1990b).

Reducing conditionsAn important factor in pink color formation is the oxidation-reduc-

tion potential (ORP) which determines the state of thehemochrome(Fe2+)/hemichrome(Fe3+) iron of globin hemochromesand is the determining factor in the ability of pigments to combinewith or tie up molecules. The ORP is a function of the pH and thepresence of reductants. The high reducing conditions allow greaterreactivity and interconversion of pigments that is not desirable fromthe point of meat color (Cornforth and others 1986; Ahn and Maur-er 1989b, 1990b). The pink color intensity of the heme complexesformed from myoglobin and hemoglobin with naturally presentligands increases tremendously under reducing conditions (Ahnand Maurer 1990a). The oxidation-reduction potential tends to be-come more negative (reducing conditions) at a high pH (Antonini andBrunori 1971). The heme pigments can be reduced by a number of

reductants, some of which are endogenous to muscle tissue. In thefresh meat, the reducing substances endogenous to the tissue con-stantly reduce the oxidized pigment (Fox 1966). Meat reducing activ-ity is related to the presence of intracellular nicotinamide coenzymessuch as nicotinamide adenine dinucleotide (NAD+, reduced formNADH) and nicotinamide adenine dinucleotide phosphate (NADP+,reduced form NADPH) (Giddings 1974, 1977; Livingston and Brown1981; Renerre 1990). The nicotinamide coenzymes (also known aspyridine nucleotides) are electron carriers. They play a vital role in avariety of enzyme catalyzed oxidation-reduction reactions. It is nec-essary to maintain mildly oxidizing conditions to prevent pink colordefect in cooked meat under commercial conditions (Cornforth andothers 1986; Dobson and Cornforth 1992). The in situ levels of reduc-tants are important in controlling rates of metmyoglobin reductionto oxymyoglobin. Also, the nitrosylmetmyoglobin is formed in thepresence of reductants (for example, cysteine or ascorbic acid, sodi-um erythorbate) from nitric oxide and metmyoglobin; the nitrosyl-metmyoglobin is reduced to nitrosomyoglobin in the presence of thesame reductants (Livingston and Brown 1981). According to Fox(1966) the activity of the reductans (both ascorbate and cysteine)toward the reducing reactions depends on their ability to form a 1-electron intermediates with heme pigments. Moreover, whetherascorbate will act as an oxidant or a reductant toward myoglobindepends upon the ascorbate concentration and the presence of oth-er redox compounds. When the redox potential range is from –15 mVto –100 mV, ground cooked turkey meat has been shown to exhibit 2distinct absorption maxima, the 1st one at 542 nm and the 2nd at 580nm (Ahn and Maurer 1989b). These 2 absorption maxima are thechromatic peaks of oxymyoglobin in the 500 to 600-nm wavelengthrange. A further decrease in its potential to –550 mV reduced themyoglobin fully to the ferrous state; only 1 peak was observed at 556nm. The ORP of –550 mV or less causes heme iron to be in the ferrousstate resulting in formation of only pink reduced hemochromes(Cornforth and others 1986). The oxidation-reduction potential isalso affected by processing ingredients such as salt and phosphate.The redox changes could especially have strong effect on pinking incooked turkey breast meat in the ORP range of +90 mV to –50 mV(Ahn and Maurer 1989b). Recently, Nam and Ahn (2002a,b) reportedthat irradiation of both raw and cooked products can produce strong-ly reduced environment. Further, the vacuum packaging of irradiat-ed meat may cause additional decrease in oxidation-reduction po-tential of meat below –50 mV allowing for greater reactivity ofpotential ligands such as CO.

The role of cytochrome cAn interesting aspect is the role of cytochrome c, the electron

transport protein found in mitochondrium, in pink color defect for-mation in cooked white meat. Cytochrome c remains stable at heatlevels that myoglobin cannot withstand and is therefore thought tobe responsible for the residual pink color (Cornish and Froning 1974;Izumi and others 1982; Ahn and Maurer 1989b; Girard and others1990). Native and stable cytochrome c may retain its natural colorduring heating of meat up to 105 °C (Izumi and others 1982). Musclewith higher pH (~6.6) contains more cytochrome c than muscle withlower pH (6.0 to 6.1) (Pikul and others 1986). Chilling methods (iceslush against air chilled) may influence cytochrome c content ofchicken meat but no explanation is available (Fleming and others1991). Authors found twice as much cytochrome c in breast musclesfrom the air-chilled birds (27.15mg/g) compared with the ice-slushchilled birds (13.71mg/g). The reflectance spectra of dark raw breastmuscle from birds exposed to excitement prior slaughter and fromthose allowed to struggle freely during slaughter indicated that thedark color may be due to higher concentration of cytochrome c (Ngo-

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ka and Froning 1982). The extinction coefficient of cytochrome c at550 nm, which gives a pink red color, is about 3-fold higher than thatof myoglobin after reduction. Although the amount of myoglobin is50-fold higher than that of cytochrome c in turkey breast meat, thecontribution of cytochrome c to pinkness of breast meat could behigher than myoglobin in cooked meat (Ahn and Maurer 1989a,b).In addition, the autoxidation rate of turkey myoglobin exhibited a 6-fold slower rate in the presence of cytochrome c. The reduction ofmyoglobin is the probable cause of pinking in cooked meat since theundenatured cytochrome c remaining in meat after cooking is stillactive for electron transfer. The strong absorption peak at 414 nm,relative to other 520 nm and 550 nm remains even after cooking tur-key breast samples at 75 °C and 85 °C. In terms of proportions andpositions of the absorptions, these 3 banded spectra correspond tothe respective gamma, beta, and alpha bands, typical of cytochromec in a low ferrous state. In this form, cytochrome c appears pink (Gi-rard and others 1990). The heme complex forming reactions of cyto-chrome c are not affected greatly by pH, and the heme complexesformed by selected ligands with cytochrome c are more stable com-pared to those formed with myoglobin and hemoglobin (Ahn andMaurer 1990b). These differences may be associated with a differentstructure of cytochrome c compared to myoglobin and hemoglobin(Dickerson 1980). Moreover, degradation of mitochondria by cookingcould have facilitated release of cytochrome c from inner membraneand therefore improved its extractability for reaction (Girard andothers 1990). Lowering the redox potential from +130 mV to +30 mVcould double the absorbance of purified cytochrome c at 550 nm(Ahn and Maurer 1989b).

Efforts to prevent pinking in cooked poultry meatGood manufacturing practices concentrate on efforts to reduce or

eliminate external contamination of nitrate and nitrite such that ni-trosopigments would not significantly contribute to the pink discol-oration. Cornforth and others (1986) also proposed to create mildlyoxidized conditions in meat products during processing to preventformation of pink hemochromes after cooking. Nevertheless, theseconditions may appear to be questionable in regard to the quality ofthe final product. When studying the pinking phenomenon in whitepoultry meat researchers attempt to use pink generating ligandssuch as nicotinamide and nitrite to induce pinking (Schwarz andothers 1997, 1999; Slesinski and others 2000a,b). However, when theligands are used in combinations with other ingredients it is not clearif pinking is directly related to pink generating ligands or the inducedin situ conditions. Schwarz and others (1997, 1999) hypothesizedthat since hemochromes are formed when a specific ligand binds tothe pigment, it may be possible, without generating a pink color, tobind such a ligand to the 6th coordinate position of heme iron. Sev-eral researchers attempted to reduce pinking using different ingre-dients, such as ligands binding to heme iron, without formation ofpink color in cooked products (Schwarz and others 1997, 1999; Sles-inski and others 2000a,b). Kieffer and others (2000) showed ability of0.3% of citric acid to reduce pink defect in cooked ground turkeyproduct associated with the presence of nicotinamide and nitrosylhemochrome. However, the addition of citric acid may be of a concernto poultry producers from the point of cooking yield. In addition,there is an opportunity for some dairy proteins or some componentsof dairy proteins to reduce or eliminate the pink defect (Slesinskiand others 2000a,b); however, the mechanism responsible for the re-duction is not yet established. In addition, some of the ingredientsexamined in those studies are not yet approved to be used as non-meat ingredients in the meat or poultry products.

The mechanism of pink color reduction is not yet established.Moreover, adding the nonpink generating ligands may or may not

be effective depending on how the endogenous conditions of theraw meat were affected by processing procedures. Interactions offactors and mechanisms associated with the pink defect are com-plex. There is a need for research in the area of prediction of thepink defect based on conditions present in raw meat before cook-ing and during marination process. We believe that the simulationof the pink defect is essential as a tool to investigate procedures forits prevention and elimination. The industry has also expressed anecessity for development of models for predicting the effects ofdifferent processing conditions on discoloration in the cooked prod-ucts as evidenced by a project funded under the Georgia’s Tradi-tional Industries Program for Food Processing (GA FoodPAC 2002).

Conclusions

THE FACTORS ASSOCIATED WITH PINKING HAVE BEEN RELATED TO THE

presence of the different pigments’ classes, such as undena-tured myoglobin and oxymyoglobin, reduced ferrohemochromes,nitrosyl hemochromes, carbon monoxide hemochromes, and cyto-chrome c. The formation and chemical reactivity of these pigmentsare directly related to the in situ conditions that are represented bypH, redox potential, degree of denaturation, and presence of react-ing ligands. Regardless of which native pigments or hemochromesare involved in the pink color defect, the means to control this prob-lem have not been reported previously. Better understanding ofhow processing factors affect endogenous conditions of meat maylead to greater control over the mechanisms involved in the gener-ation of the pink defect.

ReferencesAhn DU, Maurer AJ. 1987. Concentration of nitrate and nitrite in raw turkey

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MS 20020544 Submitted 9/20/02, Revised 11/15/02, Accepted 11/26/02, Received11/26/02

Authors Holownia and Chinnan are with Dept. of Food Science and Tech-nology, Univ. of Georgia, Griffin, GA 30223. Author Reynolds is with Dept. ofFood Science and Technology-Cooperative Extension Service, Univ. of Geor-gia, Athens. Direct inquiries to author Chinnan (E-mail: chinnan@uga.edu).