Carbon Dioxide Preservation of Fresh Poultry1

Carbon Dioxide Preservation of Fresh Poultry1 C. J. WABECK 2 , C. E. PARMELEE AND W. J. STADELMAN Department of Animal Sciences, Purdue University, Lafayette, Indiana 47907 (Received for publication July 17, 1967)

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by carbon dioxide. Brown (1922) advanced the theory that inhibition of bacterial growth was based on the retarding action of carbon dioxide on the metabolic processes. Brown (1922) and King and Nagel (1965), found that a reduction of the oxygen content of the atmosphere did not limit bacterial growth. Coyne (1932) advanced the theory that inhibition was due to a change in the intracellular pH resulting from carbon dioxide penetration of the cell mass which would not result from growing an organism in an acid medium. Fromm and Monroe (1965) showed that an increase in pH, from 6.6 to 7.1 corresponded with an increase in bacterial numbers from log 4.5 to 9.5. Microbial activity appeared to play an important role in changes of skin pH. Kraft and Ayres (1961) discussed the development of fluorescence by proliferation of Pseudomonas on chicken wings. Fluorescene was not evident until counts had reached 100,000 to 1,000,000 organisms per cm2. The purpose of this study was to determine the effects of various concentrations of carbon dioxide in the atmosephere in packages of fresh poultry meat on bacterial growth. EXPERIMENTAL PROCEDURE

Coyne (1932), Haines (1933), and Scott In two experiments, the atmosphere (1938), have indicated that the two principal genera of spoilage organisms, Pseu- around each bird was controlled to deterdomonas and A chromobacter, were inhibited mine the effect of two levels of carbon dioxide on the bacterial population and the 1 Journal Paper No. 3120 of the Purdue Agricul- keeping quality. Treatments consisted of tural Experiment Station. This work was supported atmospheres of 10% and 20% carbon diin part by a grant from Anderson Box Company, oxide and an atmosphere of air. EviscerIndianapolis, Indiana. 2 ated birds were placed in individual beakPresent address: Armour and Company, Oak ers and placed in 15.1 liter aluminum Brook, Illinois. 468

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HE use of carbon dioxide as a preservative for fresh poultry was advocated as early as 1934. Smith (1934) flowed carbon dioxide into an airtight room containing non-eviscerated birds and found that at least 70% carbon dioxide was needed to hold down bacterial counts for four months at a temperature of 30°F. Smith concluded that better results were obtained in air, because evidence of green decomposition appeared on birds in carbon dioxide after eight weeks. Lea (1934) concluded that although carbon dioxide practically eliminated mold and bacterial spoilage on chickens held at 0°C, autolysis of the tissues by enzymes prevented any great extension of storage life. Eviscerated poultry held in atmospheres containing more than 20% carbon dioxide lost its bloom and had yellow skin according to Ogilvy and Ayres (1951). Storage life under atmospheric conditions, at 32°F., was 15 days. The storage period was extended to 25 days when 10% carbon dioxide, or to 45 days when 20% carbon dioxide was maintained in the atmosphere throughout storage. Reduction of storage temperature, or increase in carbon dioxide concentration, increased storage life.

CARBON D I O X I D E PRESERVATION

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containers (Figure 1). Dow Corning Silicone grease was placed on the lip around the periphery of the container. A plexiglass cover, with a hole in the center, was placed on each container and clamped in four equal positions with C-type clamps. A rubber stopper containing a long piece of glass tubing was placed in the hole in the center and the glass tube was pushed down until the end was under the surface of water in a 250 ml. beaker at the center of the bottom of the container. Atmospheres were provided from tanks of compressed air, or 10% and 2 0 % carbon dioxide balanced with air. T a n k s containing the carbon dioxide atmospheres were purchased from Matheson Co., Joliet, Illinois. T h e atmospheres provided by each tank were analyzed by Matheson for accuracy. The drip collection method of Mercuri and Kotula (1964) was used for bacterial

counts. Total bacterial counts were made by the agar plate method of the A P H A (1960) with incubation at 3 2 ° C , and with the exception t h a t . 1 % peptone rather than phosphate buffer was used for dilution. Coliform counts were made by the plate method of the A P H A (1960) using violet red bile agar and incubation at 37°C. for 24 hours. Typical dark red colonies, at least .5 mm. in diameter, were counted and confirmed by gas production in brilliant green lactose bile broth. Psychrophilic counts were made by the agar plate method described by the A P H A (1960) for butter. Lipolytic counts were made by a modification of the method of Parfitt el al. (1933) in which 2.5 ml. rather than 5 ml. of nile blue sulfate was added to each 100 ml. of agar. Estimates of p H on the skin and in the drip were made in the second experiment. The d a t a were analyzed by the orthogonal polynomial

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FIG. 1. A 15.1 liter container for storage of carcasses in atmospheres of air, 10 percent carbon dioxide or 20 percent carbon dioxide.

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C. J . W A B E C K , C. E . P A R M E L E E AND W. J . STADELMAN

Birds were held a t a temperature of 1°C. in all experiments, except for the second experiment involving all atmospheres, when the temperatures increased to 2°C. from the second to the fourth day.

organisms were in drip from birds stored in air, next largest from those stored in 10% carbon dioxide, and least from those stored in 2 0 % carbon dioxide. Inhibition b y carbon dioxide was not evident until a population of 106 organisms was reached. At the termination of the first experiment, the total count for the birds held in 10% carbon dioxide was reduced one log scale and t h a t for those held in 2 0 % carbon dioxide three log scales less than t h a t for birds stored in an atmosphere of air (Table 1). T h e change in total bacterial count on birds stored in air followed a normal pattern, whereas an irregular p a t t e r n of change was observed for total bacterial count on birds in 1 0 % a n d 2 0 % carbon dioxide atmosphere. T h e analysis of variance showed a significant linear (L) treatment (T) effect. Significant cubic (C), and highly significant linear, quadratic (Q) and remainder (R) effects were computed for days ( D ) . A highly significant effect for T L D L and T Q D C , and significant effect for T L D Q were found.

RESULTS AND DISCUSSION

Total counts are nearly the same for 6 days in experiments using the three atmospheres as shown in Table 1. At the end of the storage time, the largest numbers of

T h e irregularity of growth of t h e bacterial populations in carbon dioxide atmospheres contributed to the deviation away from linear growth for all treatments. Differences between treatments

TABLE 1.—Logarithmic bacterial counts of drip from birds stored at 1°C. 10% and 20% carbon dioxide. Experiment 1

• air,

Bacterial count (log; average)

Storage time (days)

Total

Coliform

Psychrophilic

Period

Air

10% C0 2

20% C0 2

Air

10% C0 2

20% C0 2

Air

10% C0 2

20% C0 2

0 2 4 6 8 10 12 14 16

5.26" 5.23 5.13 4.93 5.96 6.33 7.94 9.17 9.40

5.55 5.53 5.97 5.18 6.46 6.08 6.60 7.70 8.17

5.29 5.27 5.12 4.86 5.95 5.62 5.85 5.65 6.14

3.27 3.05 3.47 4.82 4.79 7.21 8.10 9.21 10.08

3.26 3.42 4.12 4.76 5.06 5.68 7.41 8.50 9.15

3.02 2.77 3.87 4.68 4.52 4.90 5.34 5.67 6.20

2.78 2.29 2.56 2.33 2.30 2.15 2.72 2.80 3.28

3.12 3.07 2.31 2.50 2.74 3.33 3.98 4.89 5.52

3.25 2.72 2.47 2.84 2.73 2.75 3.40 3.44 3.21

8

Average of four samples.

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method for analysis of variance described b y Steel and Torrie (1960). A third experiment was designed to determine whether sampling of accumulated drip was confounded b y the addition of drip from each bird every two days to t h a t accumulating in the bottom of the beaker. Mercuri and Kotula (1964) withdrew drip fluid from packages periodically for bacterial count determinations and compared the results to counts from drip fluid initially isolated from the packages and sampled at the same intervals. I n this study, total counts were made on the isolated initial drip and drip which had accumulated every two days in beakers containing birds held in the 10% carbon dioxide atmosphere. Beakers containing the isolated initial drip were also held in the 10% carbon dioxide atmosphere.

CARBON D I O X I D E P R E S E R V A T I O N

T L D R , T Q D L , and T Q D Q interactions were

found. Populations means for coliform counts were highest for 2 0 % carbon dioxide and lowest for air atmospheres (Table 1). Coliform populations were largest for the 10% and 2 0 % carbon dioxide atmosphere initially, and remained highest throughout the storage. All means remained fairly constant throughout storage, except for populations in 1 0 % carbon dioxide which increased after the eighth day. T h e analysis of variance for coliform counts showed a significant quadratic effect for treatments, and a highly significant linear and quadratic effect for days. T h e analysis for T Q D L and T Q D Q was highly significant.

The significant results m a y be due to the increase of the population in the 1 0 % carbon dioxide atmosphere while the other two populations remained stationary. Off-odor as determined by two persons was evident for birds in atmospheres of 10% and 2 0 % carbon dioxide on the tenth

day, and for birds in air on the sixteenth day. Off-odors for birds in carbon dioxide were "sweet" or "fruity," compared to " s h a r p " or " s o u r " odors for birds in an atmosphere of air. T h e off-odor of birds in carbon dioxide atmosphere was less objectionable t h a n t h a t of birds stored in air. Difference in off-odor indicated t h a t carbon dioxide had permitted t h e growth of an organism which is normally suppressed in storage b y predominating spoilage organisms. Although t h e growth of total populations was suppressed by carbon dioxide, the early off-odor resulted in an undesirable product. Birds in atmospheres of 1 0 % and 2 0 % carbon dioxide showed no evidence of fluorescence under UV light. Birds stored in air showed a gradual increase in number of fluorescent colonies from the t e n t h to the sixteenth days. Greatest number of such colonies were in areas around the tail and legs, and under the wings. Birds showed no evidence of skin discoloration, in any of the atmospheres. Carbon dioxide m a y have inhibited pigment production, or more likely, inhibited fluorescing pseudomonad populations sufficiently, so t h a t populations did not reach a minimum of 100,000 organisms per cm. 2 , reported b y Kraft and Ayres (1961) to be necessary for evidence of fluorescence under UV light. I n the second experiment, lipolytic and proteolytic counts, and p H measurements of the skin and drip were made, in addition to total, psychrophilic, and coliform counts. Total counts showed the same p a t t e r n of change observed in the first experiment except t h a t population increases in the three atmospheres were sharper, and did not exhibit the irregularity shown b y populations in carbon dioxide in the first experiment. (Table 2). T h e storage period was reduced from sixteen days to fourteen days. T r e a t m e n t linear effect was highly

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were consistent throughout storage as shown by the significant linear effect. Psychrophilic counts showed the same p a t t e r n of change which was observed for total counts in the three atmospheres (Table 1). T h e higher counts of psychrophiles than of total count after ten days or more of storage indicates a strong temperature effect on growth of types present. After the extended storage, the organisms t h a t grew at low temperatures on the birds did not survive the 32°C. temperature use for total counts. Inhibition of a portion of the population, in atmospheres of 1 0 % and 2 0 % carbon dioxide was observed from the eighth day to the end of the experiment. A highly significant linear effect was found for treatments. Linear, quadratic, cubic, and remainder effects were highly significant for days of storage. Highly significant T L D L , T L D Q , T L D C ,

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C. J. W A B E C K , C. E. P A R M E L E E AND W. J. STADELMAN

TABLE 2.—Average logarithmic bacterial counts of drip from birds stored at 1°C. 10% and 20% carbon dioxide. Experiment 2 Bacterial count (log:average)a Total Storage time (days)

10% CO2 Air

0 2 4 6 8 10 12 14 a

4.52 a 4.46 4.49 6.26 8.74 11.18 9.50 9.95

4.20 4.19 4.19 4.24 6.52 10.37 9.64 8.13

20% C02

4.47 4.27 4.24 4.23 4.85 8.46 7.25 7.16

Psychrophilic

1Colilorm

10% CO2

10% CO2

20% CO2

Air

3.52 3.13 4.10 5.78 7.19 9.05 9.15 9.51

Lipolyt:ic 20% CO2

Air

3.15 2.96 3.06 4.15 5.12 6.48 7.07 7.88

3.25 2.98 3.04 3.72 4.40 6.10 6.34 6.77

10% CO2 Air

2.11 2.47 2.55 2.04 2.39 1.75 1.73 1.60 2.39 2.13 2.03 2.12 2.06 1.72 2.19 1.85

2.55 2.09 1.69 2.49 2.09 1.99 2.20 1.95

3.95 3.48 4.01 5.57 7.25 8.19 8.75 9.30

20% C02

3.90 4.04 3.62 3.67 3.02 3.32 3.85 3.75 4.94 4.42 6.06 5.11 6.53 5.64 7.14 6.13

Proteolytic 10% CO2

20% CO2

Air

2.57 2.73 3.14 3.86 7.14 7.37 7.98 8.56

3.22 2.52 2.93 2.74 2.74 2.98 2.94 2.65 4.92 4.74 4.81 4.23 5.43 4.24 6.74 5.62

Average of three samples.

Coliform counts exhibited the same pattern observed in t h e first experiment (Table 2), with slight increases and decreases throughout storage. T h e populations on birds in the 1 0 % carbon dioxide experiment did not show the increase observed after the eighth d a y in the first experiment. Population differences were a fraction of a base logarithm throughout storage. T h e analysis of variance showed a significant effect for T Q D R . Michener and Elliott (1964) stated t h a t most conforms did not grow below 5°C. T h e results of the two experiments are in agreement. Lipolytic organisms showed the same growth patterns observed for psychrophilic organisms, with a two-day lay

phase for populations in air, and a fourday lag phase for populations on birds in 10% and 2 0 % carbon dioxide atmospheres. Populations under carbon dioxide atmospheres began to ascend on the sixth day. T h e analysis of variance showed highly significant linear and quadratic treatment, and linear, quadratic, cubic and remainder storage time effects. Highly significant effects were found for T L D L , T L D Q , T L D C , T Q D L , and T Q D Q in-

teractions. Proteolytic counts indicated a closer growth pattern to total counts. Lag phase was through the sixth d a y for populations under carbon dioxide, and the fourth d a y for populations in an atmosphere of air, followed b y a rapid increase in growth. T h e analysis of variance showed highly significant linear and quadratic effects for treatments, and a highly significant linear, cubic, and remainder effect for days. Highly significant effects were shown for T L D L , T L D C , T C D L , and T Q D Q . A group

of lipolytic and proteolytic organisms able to grow under psychrophilic conditions were inhibited b y both levels of carbon dioxide. D a y to d a y variations in counts were quite variable. An ascendency and decendency of different organisms, as a result of different initial populations, may have caused the variations. Measurement of skin p H with a Beck-

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significant. Linear, cubic, and remainder effects were highly significant for days of storage. A highly significant T L D L effect was shown. Psychrophilic counts followed the same p a t t e r n as total counts, except t h a t the lag phase of the psychrophilic organisms was approximately two days less t h a n shown b y total count (Table 2). T h e analysis of variance was highly significant for linear and quadratic t r e a t m e n t effects. A highly significant linear, cubic, and remainder effect was shown for days. Highly significant T L D L , T L D Q , T L D L , and significant T Q D Q effects were found in the T X D analysis.

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CARBON DIOXIDE PRESERVATION

TABLE 3.—Average of values of skin and drip from birds stored at 1°C. in air, 10% and 20% carbon dioxide. Experiment 2 Storage time

Skin Air

0 2 4 6 8 10 12 14 a

6.76" 6.39 6.37 6.39 6.45 6.61 6.73 6.73

Drip

10% C0 2 20% C0 2 6.69 6.39 6.13 6.41 6.24 6.20 6.36 6.29

6.75 6.42 6.35 6.59 6.39 6.42 6.49 6.24

Average for three samples.

Air 6.88 6.31 6.18 6.18 6.16 6.17 6.43 6.31

10% COa 20% COs 6.78 6.50 6.20 6.42 6.27 6.27 6.29 6.28

6.91 6.42 6.19 6.30 6.23 6.27 6.29 6.25

TABLE 4.—Average logarithmic total bacterial count from isolated initial and fresh two day drip of .birds in 10% carbon dioxide stored at 1°C. Experiment 3 Total bacterial count (log average) (days) 2 4 6 8 10 12 14 16 18 a

Initial drip

Fresh drip

5.04a 4.83 4.27 4.44 4.81 4.94 6.01 6.94 8.23

5.04 4.89 4.75 4.94 5.00 5.11 6.00 6.73 8.27

Average of four samples.

air were putrid on the fourteenth day. No evidence of discoloration on the skin was observed. The third experiment conducted with birds under the 10% carbon dioxide atmosphere showed no apparent differences in total bacterial counts between the original drip and fresh drip (Table 4). The comparative study of isolated initial drip and that collected every two days showed that a sample of the drip taken initially supported growth of the organisms away from the bird as successfully as on the bird during storage, which is in agreement with the findings of Mercuri and Kotula (1965). Samples of drip taken in the processing plant could be used as an index of sanitation, and to estimate storage life without loss of birds. Off-odor was evident on the twelfth day. The off-odor did not achieve the intensity exhibited by birds in previous experiments. Data were not analyzed statistically. Bacterial populations between birds, and experiments, as a result of the initial flora present, may have accounted for the differences observed. Difference in predominant types of organisms were observed for birds in the different atmospheres and between treatments. The ir-

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man surface electrode showed initial values to range between 6.69 and 6.76, followed by a sharp decrease in values for birds in the three atmospheres (Table 3). The values for birds in the atmosphere of air showed a gradual increase in pH which corresponded to the increase in population, whereas pH of birds in both levels of carbon dioxide increased and decreased throughout storage. Difference in pH among birds stored in the three atmospheres were not significant. The analysis of variance was highly significant for quadratic, and cubic day effects, with highly significant T L D L and significant TLDQ effects. A two-day lag effect was observed for coliforms, which corresponded to the lower skin pH at the second day, especially under 20% carbon dioxide. Drip fluid pH was measured throughout storage (Table 3). Values for pH showed the sharp decrease for the first two days followed by increases and decreases of pH values, for drip from birds in each atmosphere, throughout storage. The analysis of variance had highly significant linear, quadratic, cubic, and remainder effects for days, and a significant TQDQ effect. Off-odors occurred for the carbon dioxide treated birds by the twelfth day. One bird in the atmosphere of air had a slight off-odor on the twelfth day. All birds in

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C. J. WABECK, C. E. PAKMELEE AND W. J. STADELMAN

regularity of bacterial growth for birds in carbon dioxide may have been due to individual variations between birds, or the ascendency and descendency of bacterial species affected by carbon dioxide. SUMMARY AND CONCLUSIONS

REFERENCES American Public Health Association, 1960. Standard Methods for the Examination of Dairy Products. 11th ed. American Public Health Association, Inc., 1790 Broadway, New York 19, N. Y. p. 47-82, 121-141, 148, 209. Brown, W., 1922. On the germination and growth of fungi at various temperatures and in various concentrations of oxygen and carbon dioxide. Ann. Botany (London), 36: 257-283.

SEPTEMBER 8-12. INTERNATIONAL POULTRY INDUSTRY EXPOSITION, POULTRY INDUSTRY MANUFACTURERS' COUNCIL, INTERNATIONAL AMPHITHEATRE, CHICAGO, ILLINOIS.

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Results of Experiments 1 and 2 indicated that administration of carbon dioxide throughout storage, at 1°C, had an inhibitory effect on part of the bacterial population. Coliform populations were relatively stable during storage, due to the low temperature, but some lipolytic and proteolytic psychrophiles were suppressed by carbon dioxide. Differences in pH of the skin were statistically insignificant, although values for birds in carbon dioxide were lower than value for those in air, as measured in Experiment 2. Drip fluid pH was not consistent with skin pH, in values and treatment order. Variation of results can be attributed to differences of the mixed flora present initially, and during each experiment. Growth patterns of organisms on birds in air were normal, whereas those on birds in carbon dioxide atmospheres indicated selective inhibition. Fluorescent bacteria were not observed on birds in carbon dioxide atmosphere.

Coyne, F. P., 1932. The effect of carbon dioxide on bacterial growth with special reference to the preservation of fish. J. Soc. Chem. Ind. 50: 119T-121T. Fromm, D., and R. J. Monroe, 1965. Some observations concerning the hydrogen ion variability of the chicken carcass surface during storage. Poultry Sci. 44: 325-336. Haines, R. B., 1933. The influence of carbon dioxide on the rate of multiplication of certain bacteria. J. Soc. Chem. Ind. 52: 13T-17T. King, A. D., Jr., and C. W. Nagel, 1965. Inhibition of growth of a pseudomonas by carbon dioxide. 65th Ann. Bacterial Pro. p. 32. Kraft, A. A., and J. C. Ayres, 1961. Production of fluorescence on packaged chicken. Appl. Microbiol. p: 549-553. Lea, C. H., 1934. The cold storage of poultry. Part II. Chemical changes in the fat of gas-stored chickens. J. Soc. Chem. Ind. 53: 347T-349T. Michener, H. E., and R. P. Elliott, 1964. Minimum growth temperatures for food poisoning, fecal indicator, and psychrophilic organisms. In Chichester, C. O., ed., Advances in Food Res. 13:349-396. Mercuri, A. J., and A. W. Kotula, 1964. Relations of "Breast Swab" to "Drip" bacterial counts in tray packed chicken fryers. J. Food Sci. 29: 854-858. Ogilvy, W. S., and J. C. Ayres, 1951. Post-mortem changes in stored meats. II. The effect of atmospheres containing carbon dioxide in prolonging the storage life of cut-up chicken. Food Technol. 5:97-102. Parfitt, E. H., E. G. Wood, B. W. Hammer, H. Macey and R. S. Breed, 1933. Direction of lipolytic organisms. J. Dairy Sci. 16: 294. Scott, W. J., 1938. The growth of microorganisms on ox muscle. III. The influence of 10% carbon dioxide on rate of growth at — 1°C. Australia, Commonwealth Council Sci. Ind. Research, 11: 266-267. Smith, E. C , 1934. The cold storage of poultry. Part I. Gas storage of chickens. J. Soc. Chem. Ind. 53: 345T-347T. Steel, R. G. O., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Company, Inc., New York, N. Y. p. 222-223.