Color of Poultry Meat as Influenced by Dietary Nitrates and Nitrites1

Color of Poultry Meat as Influenced by Dietary Nitrates and Nitrites 1 G. W. F R O N I N G , J. DADDARIO, T. E. H A R T U N G , T. W. SULLIVAN AND R. M. H I L L Departments of Poultry Science and Biochemistry and Nutrition, University of Nebraska, Lincoln, Nebraska 68503 (Received for publication September 17, 1968)

I T R A T E S and nitrites have been used for some time in curing of red meat products. Nitrite reacts with myoglobin to produce nitrosomyoglobin which is red in color. Upon heating this nitrosomyoglobin is converted to a pink component nitrosohemochrome. T h e sequence in production of the cured meat color is as follows (American M e a t Institute Foundation, 1960): bacterial N03

> N02 action

N02 -

p H 5.4 to 6.0 + H ± — > HN02 reduction

HNO2

> NO

NO + myoglobin ^-nitrosomyoglobin (red) Nitrosomyoglobin heat > nitrosochemochrome (Pink) N i t r a t e in the curing mixture is ultimately reduced to nitrite by nonpathogenic organisms. The current practice is to use both nitrates and nitrites in meat curing, since nitrite alone does not produce a desirable color. If nitrite is used alone, the concentration of nitrous acid is of such magnitude t h a t it decomposes and N2O3 is lost in the process. T h e cured color reaction is desirable in m a n y products, b u t the occurrence in conventionally cooked poultry meat m a y 1 Published with the approval of the Director as Paper No. 2433, Journal Series, Nebraska Agricultural Experiment Station.

offer a possible source of undesirable pink color in certain products. T h e introduction of nitrates or nitrites into the meat m a y be possible in at least two different ways. One is the addition through the water supply in the processing plant. This is possible since water supplies may contain high nitrate levels and absorption in the slush ice chill t a n k may be sufficient to cause a pink color in the cooked meat. However, this manner of introduction would probably produce only a surface reddening. Another means of introducing nitrates or nitrites into the meat tissue is through feed or water supplies during production of the bird. Davidson (1966) reported t h a t corn, oats, various other small grains, sudan grass and sorghums m a y accumulate large quantities of nitrates, especially during hot weather and drought. M a r r e t t and Sunde (1968) found nitrate levels of 1177, 1564, 472 and 154 p a r t s per million (p.p.m.) at various times in unsupplemented basal diets for poults and chicks. Sell et al. (1963) fed two week-old chicks 0.4% nitrite in the form of potassium nitrite. There was a rapid increase in plasma nitrite following nitrite ingestion. Other workers have reported the effect of dietary nitrates and nitrites on growth rate (Adams et al., 1965, 1966, 1967; Bentley et al., 1965; Kienholz et al., 1965). Apparently none of these studies concerned the effect of dietary nitrates or nitrites on meat color. Thus, this study was designed to ascertain the color attri-

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N

POULTRY MEAT COLOR

butes of poultry meat from birds fed nitrates and nitrites. PROCEDURE

TABLE 1.—Composition of basal diet for experiment 1 Ingredient

Ground yellow corn Soybean meal (50% protein) Meat and bone scraps (50% protein) Fish meal (60% protein) Dehy. alfalfa meal (17% protein) Dried whey Dicalcium phosphate Ground limestone Salt Trace mineral mix1 Vitamin pre-mix2 Animal fat

Per kilogram diet Grams 583.0 242.0 25.0 25.0 50.0 25.0 10.0 5.0 4.0 0.5 10.0 20.0

1 Trace mineral mix provides the following per kilogram of diet: 44 mg. manganese, 44 mg. iron, 4.4 mg. copper, 1.5 mg. iodine, and 55 mg. zinc. 2 Vitamin premix provides the following per kilogram of diet: 3,300 U.S.P. units stabilized vitamin A, 8801.C. units of vitamin D 3 ,111.U. vitamin E, 22 mg. niacin, 6.6 mg. calcium pantothenate, 3.3 mg. riboflavin, 8.8 meg. vitamin Bi 2 , 330 mg. choline, 11 mg. antibiotic (Zn-bacitracin) and 1 gm. methionine-hydroxy-analog.

vac bags, frozen at — 29°C. and stored at — 29°C. until ready for testing. When ready for color evaluation, the carcasses were removed from frozen storage and thawed overnight at a refrigerated temperature of 5°C. Raw meat samples (breast and thigh meat) were taken from one half of the carcass and the other half was cooked to an internal temperature of 82.5°C. One half of the samples were cooked in aluminum foil; the remainder were cooked in open pans to internal temperature of 82.5°C. All cooking was accomplished in an electric rotary oven. Gardiner ai, values were determined on cooked (held overnight at 5°C. prior to measurement) and uncooked samples using the Gardiner Model C-4 color difference meter. Visual scores were observed on cooked (hot and cold) and uncooked samples. A visual scoring system of 1 to 5 was utilized with 1 denoting normal color and 5 extremely pink. Analysis of nitrites was accomplished using the A.O.A.C. method (23.016) for cured meats. Nitrate levels in meat samples were determined using the phenoldisulfonic acid method (A.O.A.C. 31.005). Experiment 2.-—A total of 70 Broad Breasted White turkeys were allocated equally to each of five treatments at dayold. Sodium nitrate levels were added to the basal diet to provide 0, 300, and 600 p.p.m. of nitrate; these levels were designated treatments A2, B2, and C2, respectively. Nitrites in the form of sodium nitrite were incorporated into diets at levels of 100 and 200 p.p.m., which represented treatments D2 and E2 respectively. Basal diets used throughout this study are shown in Table 2. Turkeys were fed the appropriate experimental diet and nitrate-free tap water ad libitum to fourteen weeks of age. Birds were processed using a sub-scald temperature of 60° C. following the growth period. Carcasses re-

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Experiment 1—Commercial crossbred broilers were fed sodium nitrate levels to provide 0, 150, 300, and 600 p.p.m. of added nitrate; these levels represented treatments A, B, C, and D, respectively. Nitrites were fed in form of sodium nitrite at levels of 25 and 50 p.p.m., which were designated treatments E and F respectively. The basal diet employed is shown in Table 1. This basal diet was analyzed and no nitrate was detected. Twenty birds were assigned at random to each treatment and divided into replicates of 10 birds each. Birds were fed the appropriate experimental diet and nitrate-free tap water ad libitum from day-old to nine weeks of age. Following the nine-week growth period, broilers were processed using a semi-scald temperature of 54°C. Ready-to-cook carcasses were packaged in evacuated Cryo-

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F R O N I N G , D A D D A R I O , H A R T U N G , SULLIVAN AND

TABLE 2.—Composition of basal diets for experiment 2

TABLE 3.—Nitrate analyses of chicken '•• meat tissue

Per kilogram diet Ingredient

Treatment

28% protein 0-6 weeks

25% protein 6-10 weeks

21% protein 10-14 weeks

Grams 443.0 381.0

Grams 517.0 308.0

Grams 622.1 210.0

50.0 25.0

50.0 25.0

50.0 25.0

25.0 25.0 12.5 5.0 4.0 0.8* 8.0** 0.5 3.Of 20.0

25.0 25.0 10.0 5.0 4.0 0.6 6.0 0.3 2.0 23.5 2.0

25.0 25.0 7.5

—

— 4.0 0.6 6.0 0.2 2.0 24.0 2.0

* Trace mineral mix provides the following per kilogram of diet: 4.4 mg. copper, 44.1 mg. iron; 44.1 mg. manganese, 70.5 mg. zinc and .9 mg. iodine. ** Vitamin premix provides the following per kilogram of diet: 8,800 U.S.P. units stabilized vitamin A; 2,200 I.C. units vitamin D3; 8.8 I.U. vitamin E; 3.5 mg. menadione sodium bisulfite; 7.0 mg. riboflavin; 14.1 mg. calcium pantothenate; 70.5 mg. niacin; 0.5 mg. folic acid; 14.1 meg. vitamin B12; 880 mg. choline chloride. t Antibiotic supplement provides 33 mg. zinc-bacitracin per kilogram diet.

tained for color evaluation were packaged, stored, handled and cooked as explained in Experiment 1. After cooked carcasses were evaluated, they were packaged in evacuated Cryovac bags, frozen and stored six months at — 29°C. Visual scores and Gardiner aL values were determined on the cooked samples after storage. All results were evaluated statistically using analysis of variance (Snedecor, 1957). Statistical significance is reported at the 0.05 level of probability. RESULTS AND DISCUSSION Experiment 1.—Nitrite analyses indicated no nitrite in the chicken meat samples. T h e zero p.p.m. nitrate level (Table 3) showed some nitrate. This m a y have been caused by some color interference from colored components of the tissue. Nevertheless, relative values with respect to the control appear realistic. The nitrate level in meat from birds fed 600 p.p.m. nitrate was slightly increased.

Nitrate N p.p.m. 13 13 10 27

A B C D

Higher levels of dietary nitrates significantly increased Gardiner aL values (increasing aL values denote more pinkness) of uncooked white chicken meat (Table 4). Supplemental nitrites did not significantly increase aL values of the uncooked white meat. D a r k chicken meat aL values were not affected b y dietary nitrates or nitrites. Open cooked dark meat from birds fed dietary nitrites appeared to have higher aL values than the control meat, but statistical analyses showed this trend not to be significant. Gardiner aL values of white meat from birds fed 600 p.p.m. nitrate and cooked in aluminum foil were significantly higher t h a n readings on control meat. Other trends in cooked chicken were not evident. Visual scores (Table 5) of uncooked white chicken meat were significantly increased by the three levels of dietary nitrates, whereas visual scores of uncooked dark meat were not significantly influenced by dietary nitrates of nitrites. TABLE 4.—Effect of dietary nitrates and nitrites on Gardiner ar, values of chicken meat Uncooked Treatment

A B C D E F

Open cooked

Cooked with aluminum foil wrap

White meat

Dark meat

White meat

Dark meat

White meat

Dark meat

8.5 11.5* 11.0* 10.5* 8.6 9.2

18.2 19.8 16.6 17.8 16.3 17.5

5.6 4.8 5.4 4.9 4.8 4.4

5.3 5.5 5.4 5.4 5.9 6.3

4.5 5.9 5.1 6.0* 4.3 4.3

6.1 6.4 6.4 6.1 5.6 6.2

* Significantly different than the control at .05 level of probability.

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Ground yellow corn Soybean meal (50% protein) M e a t and bone scraps (50% protein) Fish meal (60% protein) Dehy. alfalfa meal (17% protein) Corn fermentation solubles Defluorinated phosphate Ground limestone Salt (NaCl) Trace mineral mix Vitamin premix Methionine-hydroxy-analog Antibiotic supplement Animal fat Emtrymix (30% dimetridazole)

HILL

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POULTRY MEAT COLOR TABLE 5.—Effect

of dietary nitrates and nitrites on visual color scores of chicken meat

Treatment

A B C D E F

White meat

Dark meat

1.3 1.8* 1.7* 1.8* 1.2 1.1

1.5 1.7 1.7 1.7 1.3 1.6

Cooked with aluminum foil wrap

Open Cooked

Uncooked

White meat H

1

C

1

1.1 1.1 1.2 1.2 1.2 1.2

1.1 1.0 1.1 1.1 1.1 1.0

White meat

Dark meat H

l

1.0 1.0 1.0 1.1 1.1 1.0

C

1

H

1.2 1.1 1.2 1.2 1.2 1.3

1

C

1

1.1 1.0 1.0 1.1 1.1 1.2

1.0 1.0 1.0 1.1 1.0 1.2

Dark meat II 1

C1

1.1 1.0 1.0 1.2 1.0 1.2

1.2 1.2 1.3 1.2 1.1 1.3

H=Hot C = Cold * Significantly different than the control at .05 level of probability.

No apparent trends were observed with respect to visual scores of cooked meat. Experiment Z.—Nitrate and nitrite analyses of turkey dark meat are presented in Table 6. As occurred in the chicken study, some nitrate was also detected in the control meat. Nitrate level in dark turkey tissues showed an upward trend with increasing levels of nitrate. Nitrite analyses revealed 1.0 p.p.m. nitrite N in the dark meat, while no nitrite N was detected in the control. Gardiner aL values of turkey meat from birds fed dietary nitrates or nitrites are shown in Table 7. There was a significant increase in aL values of uncooked white meat from all nitrate and nitrite treatments. Nitrates did not increase aL values of the uncooked dark turkey meat. Dark meat from turkeys fed 200 p.p.m. of dietary nitrites had significantly higher aL values than control meat. Gardiner aL values of dark meat (tur-

key and chicken) were significantly greater than those of the white meat, which is indicative of higher redness common to dark meat. This agrees with previous work of Froning el al. (1968). Open cooked turkey meat data indicated that dietary nitrites did not significantly increase aL values of the white meat (Table 7), although there appears to be an upward trend from added nitrites. Gardiner aL values of turkey dark meat were significantly higher from birds fed 600 p.p.m. nitrate and 100 p.p.m. nitrite. Although other nitrate or nitrite treatments produced increased aL values of open cooked dark meat, statistical evaluation revealed no significant differences. White and dark meat cooked in aluminum foil wrap was observed to have no significant increase in aL values due to dietary treatment. The different results TABLE 7.—Effect of dietary nitrates and nitrites on Gardiner aL values of turkey meat

TABLE 6.—Nitrate and nitrite analyses of turkey dark meat tissue

A2 B2 C2 D2 E2

Nitrate N p.p.m.

Nitrite N p.p.m.

9.6 12.0 24.0

0

— —

— —

1.0 1.0

Uncooked Treatment A2 B2 C2 D2 E2

White meat

Dark meat

24.1 27.7* 26.1* 27.1* 26.4*

35.5 36.3 35.8 37.7 39.4*

Open Cooked White meat 9.4

10.9 9.4

12.3 13.1

Cooked with aluminum foil wrap

Dark meat

White meat

Dark meat

15.6 18.9 20.8* 21.0* 19.7

12.0 10.3 12.0 14.1 11.5

15.5 17.3 18.4 12.7 18.9

* Significantly different than the control at .05 level of probability.

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FRONING, DADDARIO, HARTUNG, SULLIVAN AND H I L L TABLE 8.—Effect

of dietary nitrates and nitrites on visual color scores of turkey

Treatment

A2 B2 C2 D2 E2

Cooked with aluminum foil wrap

Open cooked

Uncooked White meat

Dark meat

White meat

1.3 1.8* 1.6* 1.5* 1.7*

1.2 1.8* 1.8* 1.8* 2.0*

1.0 1.0 1.0 1.0 1.0

H

1

C

1

2.0 2.7* 2.7* 3.0* 3.0*

Dark meat H

1

1.0 1.0 1.0 1.0 1.0

C

1

2.0 2.8* 2.7* 3.4* 3.3*

Dark meat

White meat H

1

1.0 1.0 1.0 1.0 1.0

C

H1

C1

1.0 1.0 1.0 1.0 1.0

2.3 2.5* 2.7* 3.0* 2.7*

1

1.8 3.0* 2.6* 3.3* 3.6*

1

from the two cooking methods is not explainable at this time. Visual color scores of uncooked and cooked turkey meat are shown in Table 8. Visual scores of white and dark uncooked meat were increased significantly by the various nitrate and nitrite treatments. Such results, in which visual scores showed significant pinkness and not the aL values, was evident in other phases of the study to be discussed later. This contrast may be partially explained by variability in the intact muscle and difficulty in obtaining uniform sampling for at readings. The importance of a visual scoring system in conjunction with aL readings is emphasized in this study. Visual score data with both methods of cooking indicated a significant influence of dietary nitrates on color of cold samples. Hot samples showed no pinkness in any of the treatments. However, after overnight storage at refrigerated temperatures, the meat became decidedly more pink. This was true of the control also. Apparently, conversion of the brown denatured globin nicotinamide hemichrome in cooked meat is partially reversible. This conversion has been evident in some earlier work reported by this laboratory (Froning et aL, 1968). The effect of storage on meat color is shown in Table 9. Since the two cooking

methods yielded similar color trends after storage, data is presented in combined form. Six months storage largely eliminated any color differences in cooked meat. This was evident in meat from both nitrate and nitrite fed birds. Thus, the developed color, which is experienced after cooking, fades during subsequent storage at frozen temperatures. This appears to be especially noticeable in the meat from nitrite-fed birds. Although lower values of the meat from nitrite-fed birds were not shown to be significant, the results were logical in that meat from nitrate-fed birds may have greater capacity to maintain the pink color through the conversion to nitrite during storage. In general, turkey meat color was affected more by dietary nitrates and nitrites than chicken meat. Since turkey meat has a greater myoglobin concentration than chicken meat, this would appear TABLE 9.—Effect of storage at — 29°C. for six montlts on color of cooked turkey meat from birds fed various levels of dietary nitrates and nitrites Gardiner aL values

A2 B2 C2 D2 E2

Visual scores

White meat

Dark meat

White meat

Dark meat

8.2 8.2 7.3 7.8 9.5

16.1 14.6 16.0 13.2 12.2

1.5 2.0 1.8 1.9 2.2

2.3 2.2 2.3 2.0 2.2

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H=Hot C = Cold * Significantly different than the control at .05 level of probability.

POULTRY M E A T

Some color changes from dietary nitrates and nitrites were evident. These color differences from nitrates and nitrites may logically occur in field conditions since the levels used in this study approach those found occasionally in common feed ingredients ( M a r r e t t and Sunde, 1968). Although some of these color effects m a y not have apparent practical significance, they nevertheless m a y become important, if other color influencing factors in processing manifest themselves. For instance, these dietary effects might become significant in a poorly ventilated gas oven or in combination with meat from older birds.

nitrate-fed birds gave significantly higher aL values and significantly higher visual scores t h a n the white meat from control birds. Aluminum foil-cooked white chicken meat from birds fed 600 p.p.m. nitrate was observed to have significantly higher aL values than t h a t from control birds. Dietary nitrates and nitrites significantly increased visual scores of both cooked and uncooked turkey meat samples. Gardiner aL values of uncooked turkey white meat were significantly increased by dietary nitrates and nitrites. Dietary nitrites at the 200 p.p.m. level also significantly increased aL values of uncooked dark turkey meat. Open cooked dark meat from turkeys fed 600 p.p.m. nitrates or 100 p.p.m. nitrite was found to have significantly higher aL values t h a n t h a t of the control. Visual color scores of cooked chicken and turkey meat indicated virtually no pinkness in hot samples b u t after cooling pinkness was evident in all treatments including the control. There are indications of a regenerated nink color after refrigerated storage which is possibly due to a partially reversible denaturation reaction. T h e pinkness noted in meat from turkeys fed nitrites was quite evident and probably would be noted b y the consumer. Six months storage of cooked meat largely eliminated the color differences noted in the freshly cooked meat.

SUMMARY Chickens were fed nitrate levels of 0, 150, 300 and 600 p.p.m. and nitrite levels of 25 and 50 p.p.m. In the turkey phase of the study, dietary nitrates were incorporated into the diet at levels of 0, 300, and 600 p.p.m., while nitrites were added a t 100 and 200 p.p.m. M e a t samples from each of these treatments were evaluated as to color effects. Uncooked white chicken meat from

ACKNOWLEDGMENT T h e author wish to acknowledge the assistance of Mr. Delno Knudsen, Dep a r t m e n t of Agronomy, for nitrate analyses of meat samples. REFERENCES Adams, A. W., C. W. Carlson and R. J. Emerick, 1965. Effects of nitrate and nitrite in the drinking water on chicks, poults and laying hens. Poultry Sci. 44:1347.

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to be feasible. T h e inherent property of turkey meat to show considerable pinkness upon cooking is probably of significance here also. Greater levels of nitrites were also utilized in the turkey diets and the feeding period was longer, which m a y have been pertinent factors. T h e weight gain performance of birds fed dietary nitrates or nitrites was not affected in this study. However, turkeys receiving high nitrite levels were observed to be flighty and were further found to possess thick walled viscera. Hearts from these turkeys were also rather spongy a n d livers were very dark in color. If turkeys had been reared to older ages, there might have been an affect on performance from the higher nitrite levels.

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FRONING, DADDARIO, HARTUNG, SULLIVAN AND HILL Is it really a problem? Feed Age (April): 23-27. Froning, G. W., J. Daddario and T. E. Hartung, 1968. Color and myoglobin concentration in turkey meat as affected by age, sex and strain. Poultry Sci. 47: 1827. Kienholz, E. W., H. L. Enos and T. A. McPherron, 1965. The effects of some water sources and treatments upon turkey performance. Poultry Sci. 44: 1390. Marrett, L. E., and M. L. Sunde, 1968. The use of turkey poults and chickens as test animals for nitrate and nitrite toxicity. Poultry Sci. 47: 511519. Sell, J. L., W. K. Roberts and D. G. Waddell, 1963. Methemoglobinemia and reduced feed consumption due to feeding nitrite to chicks. Poultry Sci. 42: 1474-1476. Snedecor, G. W., 1957. Statistical Methods Applied to Experiments in Agriculture and Biology. Iowa State College Press, Ames, Iowa.

Studies with Corn-Soya Laying Diets 8. REQUIREMENTS FOR LIMITING AMINO ACIDS—THE BASAL DIET AND THE REQUIREMENTS FOR ISOLEUCINE, LYSINE AND TRYPTOPHAN D. J. BRAY Department of Animal Science, University of Illinois, Urbana, Illinois 61801 (Received for publication September 18, 1968)

L

IMITED experimental data are availJ able to document proposed amino acid requirement standards for laying pullets (National Research Council, 1966). The expression of requirements in terms of percent of the diet is becoming increasingly complex as new relationships among amino acids and between amino acids and other nutrients are recognized. For example, a favorable balance of amino acids tends to reduce the dietary protein requirement as demonstrated by Johnson and Fisher (1959). Since it is unlikely that the requirements for all amino acids vary in the same manner relative to the level of protein in the diet, the most useful requirement data for future refer-

ence will be those determined with amino acid-adequate diets that contain low levels of protein. In previous reports it was shown that corn-soya diets must contain 12% or less of protein to depress the performance of pullets during either long-term or shortterm assays (Bray and Morrissey, 1962; and Bray, 1968a, respectively). Corn provides about 60% and soybean meal 40% of the protein in 12% protein diets. When this protein mixture was fed at the 8.5% dietary protein level by diluting the corn and soybean meal portion of the diet with starch, essential amino acids rather than total dietary nitrogen were found to be the most limiting factor for layer perfor-

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Adams, A. W., R. J. Emerick and C. W. Carlson, 1966. Effects of nitrate and nitrite in the drinking water on chicks, poults, and laying hens. Poultry Sci. 45: 1215-1222. Adams, A. W., A. J. Kahrs and J. L. West, 1967. Effect of sodium nitrate in the drinking water on performance of turkeys. Poultry Sci. 46: 1226. American Meat Institute Foundation, 1960. The Science of Meat and Meat Products. Reinhold Publishing Corporation, New York, New York. p. 329-334. Association of Official Agricultural Chemists, 1965. Official Methods of Analysis of the Association of Official Agricultural Chemists. Tenth Edition. Published by Association of Official Agricultural Chemists. Washington, D. C. 20044. Bentley, A. B., Q. B. Kinder, G. B. Garner and J. E. Savage, 1965. Effect of nitrate in water performance of laying hens. Poultry Sci. 44: 1351. Davidson, K. L., 1966. Sublethal nitrate poisoning—