- Email: [email protected]
Spoilage Odors in Poultry Meat Produced by Pigmented and Nonpigmented Pseudomonas
QUALITY OF CARTONED AND SEALED EGGS
refrigerated immediately when Haugh unit scores were compared after one or two weeks storage at 12° C. From these results, sealing of the carton appears to be a viable alternative to immediate refrigeration of fresh shell eggs. ACKNOWLEDGEMENTS
REFERENCES Cotterill, O. J., and F. A. Gardner, 1957. Retarding thick white deterioration by holding shell eggs in sealed containers. Poultry Sci. 36: 196-206. Haugh, R. R., 1937. Haugh units for measuring egg quality. U.S. Egg Poultry Magazine, 43: 552. Marion, J. E., 1968. Wrapping eggs in film aids retention of quality. Georgia Agr. Res. 9 (4): 56. Mellor, D. B., F. A. Gardner and M. M. Miller, 1971. Interior quality decline in eggs packed in cartons warm and pre-cooled before cartoning. Poultry Sci. 50: 1605-1606. Mellor, D. B., and F. A. Gardner, 1975. An evaluation of interior quality of shell eggs packed in the carton while still warm. Poultry Sci. 54: 461-465.
Spoilage Odors in Poultry Meat Produced by Pigmented and Nonpigmented Pseudomonas N . A. Cox,
B . J. JUVEN, J. E. THOMSON, A. J. MERCURI AND V. CHEW
Animal Products Utilization and Marketing Research Laboratory, Richard B. Russell Agricultural Research Center, U.S.D.A., A.R.S., Athens, Georgia 30604 (Received for publication March 17, 1975)
ABSTRACT Twenty isolates identified as Pseudomonas were obtained from spoiled poultry carcasses. Eleven isolates were pigmented strains (P. boreopulis, P. convexa, P. fairmontensis, P. syncyanea) and the other nine were nonpigmented (P. fragi). To determine whether there was a difference in the spoilage capabilities of pigmented and nonpigmented strains of Pseudomonas, each of the 20 organisms was inoculated into four different sterilized media: (1) blended chicken breast meat and water, (2) blended chicken skin and water, (3) chicken bouillon (commercial), and (4) nutrient broth in which poultry breast meat was substituted for beef extract. Incubation was at 20° C. for 7 days or 4° C. for 14 days. Experienced judges individually evaluated coded samples for intensity and character of odor at the end of each incubation period. Statistical analysis showed that there were no differences in mean odor scores among media, between incubation temperatures or in the opinions of judges. However, difference between pigmented and nonpigmented strains was significant (p = .001); the nonpigmented strains produced more intensive "off" odors. POULTRY SCIENCE 54: 2001-2006, 1975
INTRODUCTION
SEUDOMONAS and Achromobacter (Acinetobacter) are the main spoilage organisms of chilled poultry (Ayres et al., 1950; Barnes and Shrimpton, 1958; Nagel et al., 1960; Barnes and Thornley, 1966) and are ever present in the chill waters and poultry processing plant equipment (Barnes, 1960).
P
The shelf life during chilled transport and storage is dependent on the numbers of these microorganisms that are initially present on the carcass. Poultry meat is usually considered spoiled when the microbial level reaches 106-107 cells per cm.2 of skin surface (Lochhead and Landerkin, 1935; Walker and Ayres, 1956). Barnes and Impey (1968)
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 10, 2015
Assistance with statistical analysis by Mr. James H. Denton and Mrs. Isabelle Lee is gratefully acknowledged. To simplify information, trade names of products have been used. No endorsement of named products is intended nor is criticism implied of similar products not mentioned.
2001
2002
Cox,
JUVEN, THOMSON, MERCURI AND CHEW
The objectives of the study reported here were: (1) to identify the pigmented and nonpigmented strains of Pseudomonas isolated from spoiled chicken and (2) to determine whether there are differences in the intensity of spoilage odors produced by pigmented and nonpigmented Pseudomonas in various media containing poultry constituents.
MATERIALS AND METHODS Identification of Poultry Isolates. In a previous study (Cox et al., 1974), approximately 150 Pseudomonas organisms were isolated from spoiled chicken carcasses stored at 4° C. Eighty percent of the spoilage flora of that study were identified as Pseudomonas. Twenty of these cultures were randomly selected from that group for use in the study reported here. These 20 isolates were identified as Pseudomonas according to a method described by Barnes and Thornley (1966). Species identification was made by use of Bergey's Manual of Determinative Bacteriology (Breed et al., 1957). Although the organisms had originally been identified as far as genera, some of the more important
tests were repeated to confirm the original classification. Some of the tests and media employed included: determination of morphology from a brain heart infusion (BHI) agar (Difco) slant; determination of motility by microscopic examination of a hanging drop of a 24-hr. BHI broth culture; Rhodes (1958) method for flagella stain; confirmation of fluorescence by inoculation of Pseudomonas F agar (Difco) and Pseudomonas P agar (Difco) 1 ; growth on cetrimide (pseudosel agar, Difco); determination of ability to attack cellulose in the medium of Alarie and Gray (1947) during incubation for 2 weeks; indole production (tryptone broth, BBL); nitrate reduction (trypticase nitrate broth, BBL); gelatinase activity (nutrient gelatin, Difco); oxidase reaction (oxidase discs, Difco); determination of sensitivity to penicillin and oxytetracycline by using Dispens-o-discs (Difco); determination of ability to grow at 25°, 37° and 42° C. by using BHI broth and agar (Difco); action in litmus milk (Difco); determination of the ability to hydrolyze arginine by using the method of Thornley (1960); testing of oxidative metabolism of glucose in the medium of Hugh and Leifson (1953). Selection and Composition of Spoilage Test Media. In a preliminary study, various media were formulated and tested as possible substrates for determining spoilage capabilities of pure cultures of organisms previously isolated from spoiled broilers stored in polyethylene bags at 4° C. The media were: (1) heart infusion broth (Difco); (2) sterile comminuted chicken meat (one part meat and two parts distilled water, blended in a Waring blendor and autoclaved); (3) sterile skin (one part chicken skin and two parts distilled water, blended in a Waring blendor and
1. The nonpigmented strains were repeatedly subcultured and reinoculated on these media for 2 weeks to confirm their "nonpigmented" classification.
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 10, 2015
reported that pigmented and nonpigmented strains of Pseudomonas were among the most predominant organisms growing on poultry carcasses stored at 1° C. When inoculated into minced poultry breast muscle, both the pigmented and nonpigmented strains grew very well. The nonpigmented predominated at the time of spoilage, although initially they represented only 2% of the population while the pigmented represented 35.5%. Barnes and Shrimpton (1958) stated that the growth of nonpigmented pseudomonads during storage explains the poor correlation between the development of "off" odors and the appearance of fluorescence. Fluorescence indicates presence of pigmented strains of Pseudomonas and a negative finding does not necessarily indicate a satisfactory microbiological condition.
2003
PSEUDOMONAS SPOILAGE IN POULTRY
On the basis of the preliminary tests, four of the media were found to be suitable for this experiment (numbers 3, 4,7 and 8), while the other four had masking odors. Prior to being autoclaved 20-ml. amounts of the media were dispensed in 160-ml. capacity milk dilution bottles. Inoculation of Test Media. The cultures were grown on brain heart infusion agar (Difco) slants for 18 hrs. and then washed from the medium with sterile physiological saline. The suspensions were diluted with additional saline until optical density (OD) was 0.3 at 540 nm. with a Bausch and Lomb Spectronic 20 spectrophotometer. Each of the 20 organisms was inoculated (approximately one million cells per ml. of medium) into each of the four media in duplicate. One set of the inoculated media was incubated at 20° C. for 7 days, the other at 4° C. for 14 days. Uninoculated bottles of each medium were also incubated to serve as controls. Evaluation of Spoilage Odor. Two experienced judges individually evaluated coded samples for intensity of odor at the end of each incubation period. Samples were scored
for off-odor as follows: 4—none; 3—slight; 2—definite; 1—intense as compared with the control. The statistical analysis of the data collected in our study follows that of a split-plot experimental design, where media, temperatures and strains were the main-plot treatments and opinions of judges were the sub-plot treatments, with further partitioning of the main effects into strains and their interactions with media, temperatures and opinions of judges. Growth Studies. One isolate of each species (prepared as above) was inoculated into both poultry bouillon broth and poultry nutrient broth and incubated at 20° C. The initial inoculum was approximately 1000 cells per ml. of broth. Growth patterns were determined by removing the cultures from the incubator at 0, 2, 4, 6, 8, 10, 13, 25 and 73 hours, making serial dilutions and then spreading 0.1 ml. onto brain heart infusion agar (Difco) plates with sterile bent glass rods. The plates were incubated at 20° C. for 72 hrs. RESULTS AND DISCUSSION Identification of Poultry Isolates. The identification of 20 isolates from spoiled broiler carcasses used in the study is shown in Table 1. Four different species of Pseudomonas comprised the pigmented group, while all nine nonpigmented isolates were identified as strains of P. fragi. The species nomencla-
TABLE 1.—Species identification1 of
pseudomonads from spoiled broiler carcasses Organism P. fragi (nonpigmented) P. boreopulis (pigmented) P. fairmontensis (pigmented) P. syncyanea (pigmented) P. convexa (pigmented) 'Based on Breed et al. (1957). 2 Out of 20.
Number isolated2 9 5 3 2 1
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 10, 2015
autoclaved); (4) sterile breast meat (one part broiler breast meat and two parts distilled water, blended in a Waring blendor and autoclaved); (5) unsterile breast meat (one part broiler breast meat and two parts distilled water, blended in a Waring blendor, but not autoclaved); (6) sterile "drip" (exudate or weep water collected from broilers bagged in polyethylene bags for 24 hrs. at 1 ° C ; after which the exudate was autoclaved); (7) sterile poultry bouillon broth—3 g. of chicken bouillon cubes (Armour), 5 g. peptone (Difco), 1000 ml. distilled water, heated to boiling to dissolve and then autoclaved; (8) sterile poultry nutrient broth (3 g. broiler breast meat, 5 g. peptone (Difco), 1000 ml. distilled water, heated to boiling to dissolve and then autoclaved).
2004
Cox, JUVEN, THOMSON, MERCURI AND CHEW
Odor Evaluation. A summary of the statistical analysis of the data colllected in our study is shown in Table 2. There were no
TABLE 2.—Statistical significance of mean odor scores obtained from analysis of variance Source of variation
Degrees of freedom Significance 3 n.s.
Media Temperature of incubation 1 n.s. Pigmented vs. nonpigmented' 1 ***2 Among pigmented 10 n.s. Among nonpigmented 8 n.s. Judges 1 n.s. 'Mean odor score of pigmented strains was 2.6; mean odor score of nonpigmented strains was 1.7. (Scores ranged from 1-4; 1 being intense off odor and 4 being identical to the uninoculated control). Significant at the 0.1% level.
significant differences in mean odor scores among media, between incubation temperatures or between judges. However, there was a highly significant difference (P < .001) in odors produced between the pigmented and nonpigmented strains of Pseudomonas, and this difference was independent of media, temperatures and judges. The nonpigmented strains produced much more intensive "off" odors in these media. The odors encountered in the study seemed to be identical to those described by Thornley et al. (1960). They reported that a putrid, ammoniacal odor appears when nonpigmented Pseudomonas reaches a level exceeding 10 7 /cm. 2 of skin. Growth Studies. To ascertain whether the differences in odor intensity were related to growth rates of the pigmented and nonpigmented strains, the growth patterns of the five different species of Pseudomonas were determined (Tables 3 and 4). Statistical analysis showed no significant differences in rate of multiplication among the five different species of Pseudomonas in either poultry bouillion broth or p-nutrient broth. Thus, the attainment of sufficient cell numbers per se, could not explain the differences in spoilage odor. Rey et al. (1969) found that proteinase activity of Pseudomonas isolated from chicken continued to increase even after cell counts reached their maximum. This may help explain the "ammoniacal" odors that were observed regardless of growth patterns. Our results suggest that the intensity of odor development during spoilage of poultry meat by Pseudomonas is more closely associated with the nature of the metabolism of the particular organism, than with differences in rates of multiplication among species or with various components of the poultry carcass. For example, there was no significant difference in odor intensity between medium no. 2 (chicken skin and water) and the other media tested that did not contain a great deal of chicken fat.
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 10, 2015
ture is based on the best classification criteria available at the time the study was conducted. However, with the recent publication of Bergey's Manual of Determinative Bacteriology (Buchanan and Gibbons, 1974), the following should be noted: P. boreopulis, P. fragi and P. syncyanea are still recognized as species, P. convexa is now called P. putida, and P. fairmontensis is not included in the new edition under any name and would now simply be classified as Pseudomonas sp. Nonpigmented strains of Pseudomonas have frequently been isolated from spoiled carcasses and in many instances were the predominant microorganisms on poultry spoiling at about 1° C. (Barnes and Shrimpton, 1958; Ingram and Thornley, 1959; Thornley et al., 1960; Barnes and Melton, 1971). Ayres et al. (1950) and Cox et al. (1974) isolated P. fragi along with other nonpigmented strains from spoiled broiler carcasses. Characteristics of the P. fragi isolates in our study were the same as those reported by these earlier investigators. Castell et al. (1959) identified P. fragi as the organism responsible for odors that develop during spoilage in fish muscle.
PSEVDOMONAS SPOILAGE IN POULTRY
2005
TABLE 3.—Growth of the different species of Pseudomonas in poultry bouillon broth at 20° C.
P. fairmontensis 2.94 3.04 2.53 3.69 4.11 4.54 5.08 7.85 9.18
Cultures P. syncyanea 2.79 3.08 2.84 3.65 3.61 4.49 5.23 7.77 9.30
P. boreopulis 2.97 3.15 3.36 3.89 4.23 4.90 5.53 8.11 9.30
P. convexa 2.77 2.69 3.23 3.62 4.08 4.15 4.98 7.57 8.98
'Logarithmic value of number of organisms per ml. of broth.
TABLE 4.—Growth of the different species of Pseudomonas in p-nutrient broth at 20° C. Cultures P. P. P. Time (hr.) fragi fairmontensis syncyanea 0 2.94' 2.73 2.72 2 3.00 2.83 2.67 4 3.11 2.95 2.65 6 3.43 3.36 3.18 8 3.43 3.18 3.84 10 3.73 3.86 4.00 13 4.54 4.08 4.08 25 7.90 7.08 7.60 73 9.52 9.40 9.38 'Logarithmic value of number of organisms per ml. of broth.
T h e s e results provide further evidence that nonpigmented strains of Pseudomonas a r e t h e principal microorganisms responsible for spoilage of poultry carcasses at refrigeration temperatures. ACKNOWLEDGEMENTS T h e authors are indebted to Mr. R. L . Richardson, Mr. R. H . D e a n , M s . Elizabeth Gable a n d Mr. Winston B o w e n for their technical assistance. Mention of specific b r a n d n a m e s does not imply e n d o r s e m e n t by the authors or institutions at which they are employed t o t h e exclusion of others not mentioned. REFERENCES Marie, A. M., and P. H. H. Gray, 1947. Aerobic bacteria that decompose cellulose, isolated from
P. boreopulis 2.97 2.89 3.20 3.58 3.83 4.28 4.74 7.65 9.43
P. convexa 2.65 2.76 2.93 3.32 3.89 3.90 4.48 8.26 9.49
Quebec soils. I. Isolation and description of the species. Can. J. Res. C, 25(6): 228-241. Ayres, J. C , W. S. Ogilvy and G. F. Stewart, 1950. Post-mortem changes in stored meats. 1. Microorganisms associated with development of slime on eviscerated cut-up poultry. Food Technol. 4: 199205. Barnes, E. M., 1960. Bacteriological problems in broiler preparation and storage. R. Soc. Health. J. 80: 145-148. Barnes, E. M., and C. S. Impey, 1968. Psychrophilic storage bacteria of poultry. J. Appl. Bact. 31: 97-107. Barnes, E. M., and W. Melton, 1971. Extracellular enzymic activity of poultry spoilage bacteria. J. Appl. Bact. 34(3): 599-609. Barnes, E. M., andD. H. Shrimpton, 1958. The effect of the tetracycline compounds on the storage life and microbiology of chilled eviscerated poultry. J. Appl. Bact. 21: 313-329. Barnes, E. M., andM. J. Thornley, 1966. The spoilage flora of eviscerated chickens stored at different temperatures. J. Fd. Technol. 1: 113-119. Breed, R. S., E. G. D. Murray and N. R. Smith,
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 10, 2015
Time (hr.) 0 2 4 6 8 10 13 25 73
P. fragi 3.00' 3.04 2.94 3.99 4.61 4.86 5.68 8.62 9.28
2006
Cox, JUVEN, THOMSON, MERCURI AND CHEW
investigation of bacterial action at chill temperatures. Sci. Agr. 15: 765-770. Nagel, C. W., K. L. Simpson, H. Ng, R. H. Vaughn and G. F. Stewart, 1960. Microorganisms associated with spoilage of refrigerated poultry. Food Technol. 14: 21-23. Rey, C. R., A. A. Kraft, R. G. Seals and E. W. Bird, 1969. Influence of temperature on some biochemical characteristics of Pseudomonas associated with spoilage of chicken. J. Food Sci. 34: 279-283. Rhodes, M. E., 1958. The cytology of Pseudomonas spp. as revealed by a silver-plate staining method. J. Gen. Microbiol. 18: 639-648. Thornley, M. J., 1960. The differentiation of Pseudomonas from the gram negative bacteria on the basis of arginine metabolism. J. Appl. Bact. 23: 37-52. Thornely, M. J., M. Ingram and E. M. Barnes, 1960. The effects of antibiotics and irradiation on the Pseudomonas-Achromobacter flora of chilled poultry. J. Appl. Bact. 23(3): 487-498. Walker, H. W., and J. C. Ayres, 1956. Incidence and kinds of microorganisms associated with commercially dressed poultry. Appl. Microbiol. 4: 345-349.
NEWS AND NOTES (Continued from page 1997) The Merck Company Foundation is funded by Merck & Co., Inc. to help provide financial support to many programs concerned with the needs of education, social welfare, health, medicine, science and civic betterment. Grants in 1974 were almost $2 million bringing total Foundation contributions since 1957 to nearly $19 million. COLORADO NOTES "Vitamin Deficiencies in Chickens and Turkeys," a 23 minute, 16 mm. color film for people needing information on the various symptoms caused by deficiencies of vitamin A, D, E, K, riboflavin, biotin, and folacin, is available from Colorado State University. Produced by Colorado State in association with Hoffmann-La Roche, Inc. of Nutley, New Jersey, the film is in seven distinct sections that can be tailored for showing by individual lecturers. Rental of the film is $30.00 per day, and it may be purchased for $250. Contact: Office of Educational
Media, Colorado State University, Fort Collins, Colorado 80523. Gordon E. Lacy, owner-operator, Highland Egg Ranch, Ault, Colorado, and Greeley Poultry, Greeley, Colorado, received the Distinguished Service Award Plaque at the recent Avian Science Association banquet at Colorado State University. At the fourth annual banquet of the Avian Science Club, Milton Landers received the Avian Science Club Scholarship of $250 and the $500 Gordon Lacy Scholarship. David Sharkey received the $300 Robbins Incubator Award for outstanding work in the area of embryology and hatchability, and the Jake Kellogg Memorial Scholarship, donated by Gordon Lacy, for $500. The Outstanding Graduating Avian Science Major Award was awarded jointly to Dianne Fagerberg and Jim Ranson. Dr. Carey Quarles, Professor of Avian Science and Assistant Department Head of Animal Sciences at Colorado State, was presented with a plaque recognizing his many contributions to the Club. Mr. Gordon Lacy, a Colorado egg producer and processor, was
(Continued on page 2018)
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 10, 2015
1957. Bergey's Manual of Determinative Bacteriology, 7th ed. The Williams and Wilkins Co., Baltimore, Md. Buchanan, R. E., and N. E. Gibbons, 1974. Bergey's Manual of Determinative Bacteriology, 8th ed. The Williams and Wilkins Co., Baltimore, Md. Castell, C. H., M. F. Greenough and J. Dale, 1959. The action of Pseudomonas on fish muscle. III. Identification of organisms producing fruity and oniony odors. J. Fish. Res. Bd. Can. 16: 13-19. Cox, N. A., A. J. Mercuri, B. J. Juven, J. E. Thomson and V. Chew, 1974. Evaluation of succinic acid and heat to improve the microbiological quality of poultry meat. J. Food Sci. 39: 985-987. Hugh, R., and E. Leifson, 1953. The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram-negative bacteria. J. Bact. 66: 24-26. Ingram, M., and M. J. Thornley, 1959. Changes in spoilage pattern of chicken meat as a result of irradiation. Int. J. Appl. Rad. Isotopes 6: 122-128. Lochhead, A. G., and G. B. Landerkin, 1935. Bacteriological studies of dressed poultry. I. Preliminary
refrigerated immediately when Haugh unit scores were compared after one or two weeks storage at 12° C. From these results, sealing of the carton appears to be a viable alternative to immediate refrigeration of fresh shell eggs. ACKNOWLEDGEMENTS
REFERENCES Cotterill, O. J., and F. A. Gardner, 1957. Retarding thick white deterioration by holding shell eggs in sealed containers. Poultry Sci. 36: 196-206. Haugh, R. R., 1937. Haugh units for measuring egg quality. U.S. Egg Poultry Magazine, 43: 552. Marion, J. E., 1968. Wrapping eggs in film aids retention of quality. Georgia Agr. Res. 9 (4): 56. Mellor, D. B., F. A. Gardner and M. M. Miller, 1971. Interior quality decline in eggs packed in cartons warm and pre-cooled before cartoning. Poultry Sci. 50: 1605-1606. Mellor, D. B., and F. A. Gardner, 1975. An evaluation of interior quality of shell eggs packed in the carton while still warm. Poultry Sci. 54: 461-465.
Spoilage Odors in Poultry Meat Produced by Pigmented and Nonpigmented Pseudomonas N . A. Cox,
B . J. JUVEN, J. E. THOMSON, A. J. MERCURI AND V. CHEW
Animal Products Utilization and Marketing Research Laboratory, Richard B. Russell Agricultural Research Center, U.S.D.A., A.R.S., Athens, Georgia 30604 (Received for publication March 17, 1975)
ABSTRACT Twenty isolates identified as Pseudomonas were obtained from spoiled poultry carcasses. Eleven isolates were pigmented strains (P. boreopulis, P. convexa, P. fairmontensis, P. syncyanea) and the other nine were nonpigmented (P. fragi). To determine whether there was a difference in the spoilage capabilities of pigmented and nonpigmented strains of Pseudomonas, each of the 20 organisms was inoculated into four different sterilized media: (1) blended chicken breast meat and water, (2) blended chicken skin and water, (3) chicken bouillon (commercial), and (4) nutrient broth in which poultry breast meat was substituted for beef extract. Incubation was at 20° C. for 7 days or 4° C. for 14 days. Experienced judges individually evaluated coded samples for intensity and character of odor at the end of each incubation period. Statistical analysis showed that there were no differences in mean odor scores among media, between incubation temperatures or in the opinions of judges. However, difference between pigmented and nonpigmented strains was significant (p = .001); the nonpigmented strains produced more intensive "off" odors. POULTRY SCIENCE 54: 2001-2006, 1975
INTRODUCTION
SEUDOMONAS and Achromobacter (Acinetobacter) are the main spoilage organisms of chilled poultry (Ayres et al., 1950; Barnes and Shrimpton, 1958; Nagel et al., 1960; Barnes and Thornley, 1966) and are ever present in the chill waters and poultry processing plant equipment (Barnes, 1960).
P
The shelf life during chilled transport and storage is dependent on the numbers of these microorganisms that are initially present on the carcass. Poultry meat is usually considered spoiled when the microbial level reaches 106-107 cells per cm.2 of skin surface (Lochhead and Landerkin, 1935; Walker and Ayres, 1956). Barnes and Impey (1968)
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 10, 2015
Assistance with statistical analysis by Mr. James H. Denton and Mrs. Isabelle Lee is gratefully acknowledged. To simplify information, trade names of products have been used. No endorsement of named products is intended nor is criticism implied of similar products not mentioned.
2001
2002
Cox,
JUVEN, THOMSON, MERCURI AND CHEW
The objectives of the study reported here were: (1) to identify the pigmented and nonpigmented strains of Pseudomonas isolated from spoiled chicken and (2) to determine whether there are differences in the intensity of spoilage odors produced by pigmented and nonpigmented Pseudomonas in various media containing poultry constituents.
MATERIALS AND METHODS Identification of Poultry Isolates. In a previous study (Cox et al., 1974), approximately 150 Pseudomonas organisms were isolated from spoiled chicken carcasses stored at 4° C. Eighty percent of the spoilage flora of that study were identified as Pseudomonas. Twenty of these cultures were randomly selected from that group for use in the study reported here. These 20 isolates were identified as Pseudomonas according to a method described by Barnes and Thornley (1966). Species identification was made by use of Bergey's Manual of Determinative Bacteriology (Breed et al., 1957). Although the organisms had originally been identified as far as genera, some of the more important
tests were repeated to confirm the original classification. Some of the tests and media employed included: determination of morphology from a brain heart infusion (BHI) agar (Difco) slant; determination of motility by microscopic examination of a hanging drop of a 24-hr. BHI broth culture; Rhodes (1958) method for flagella stain; confirmation of fluorescence by inoculation of Pseudomonas F agar (Difco) and Pseudomonas P agar (Difco) 1 ; growth on cetrimide (pseudosel agar, Difco); determination of ability to attack cellulose in the medium of Alarie and Gray (1947) during incubation for 2 weeks; indole production (tryptone broth, BBL); nitrate reduction (trypticase nitrate broth, BBL); gelatinase activity (nutrient gelatin, Difco); oxidase reaction (oxidase discs, Difco); determination of sensitivity to penicillin and oxytetracycline by using Dispens-o-discs (Difco); determination of ability to grow at 25°, 37° and 42° C. by using BHI broth and agar (Difco); action in litmus milk (Difco); determination of the ability to hydrolyze arginine by using the method of Thornley (1960); testing of oxidative metabolism of glucose in the medium of Hugh and Leifson (1953). Selection and Composition of Spoilage Test Media. In a preliminary study, various media were formulated and tested as possible substrates for determining spoilage capabilities of pure cultures of organisms previously isolated from spoiled broilers stored in polyethylene bags at 4° C. The media were: (1) heart infusion broth (Difco); (2) sterile comminuted chicken meat (one part meat and two parts distilled water, blended in a Waring blendor and autoclaved); (3) sterile skin (one part chicken skin and two parts distilled water, blended in a Waring blendor and
1. The nonpigmented strains were repeatedly subcultured and reinoculated on these media for 2 weeks to confirm their "nonpigmented" classification.
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 10, 2015
reported that pigmented and nonpigmented strains of Pseudomonas were among the most predominant organisms growing on poultry carcasses stored at 1° C. When inoculated into minced poultry breast muscle, both the pigmented and nonpigmented strains grew very well. The nonpigmented predominated at the time of spoilage, although initially they represented only 2% of the population while the pigmented represented 35.5%. Barnes and Shrimpton (1958) stated that the growth of nonpigmented pseudomonads during storage explains the poor correlation between the development of "off" odors and the appearance of fluorescence. Fluorescence indicates presence of pigmented strains of Pseudomonas and a negative finding does not necessarily indicate a satisfactory microbiological condition.
2003
PSEUDOMONAS SPOILAGE IN POULTRY
On the basis of the preliminary tests, four of the media were found to be suitable for this experiment (numbers 3, 4,7 and 8), while the other four had masking odors. Prior to being autoclaved 20-ml. amounts of the media were dispensed in 160-ml. capacity milk dilution bottles. Inoculation of Test Media. The cultures were grown on brain heart infusion agar (Difco) slants for 18 hrs. and then washed from the medium with sterile physiological saline. The suspensions were diluted with additional saline until optical density (OD) was 0.3 at 540 nm. with a Bausch and Lomb Spectronic 20 spectrophotometer. Each of the 20 organisms was inoculated (approximately one million cells per ml. of medium) into each of the four media in duplicate. One set of the inoculated media was incubated at 20° C. for 7 days, the other at 4° C. for 14 days. Uninoculated bottles of each medium were also incubated to serve as controls. Evaluation of Spoilage Odor. Two experienced judges individually evaluated coded samples for intensity of odor at the end of each incubation period. Samples were scored
for off-odor as follows: 4—none; 3—slight; 2—definite; 1—intense as compared with the control. The statistical analysis of the data collected in our study follows that of a split-plot experimental design, where media, temperatures and strains were the main-plot treatments and opinions of judges were the sub-plot treatments, with further partitioning of the main effects into strains and their interactions with media, temperatures and opinions of judges. Growth Studies. One isolate of each species (prepared as above) was inoculated into both poultry bouillon broth and poultry nutrient broth and incubated at 20° C. The initial inoculum was approximately 1000 cells per ml. of broth. Growth patterns were determined by removing the cultures from the incubator at 0, 2, 4, 6, 8, 10, 13, 25 and 73 hours, making serial dilutions and then spreading 0.1 ml. onto brain heart infusion agar (Difco) plates with sterile bent glass rods. The plates were incubated at 20° C. for 72 hrs. RESULTS AND DISCUSSION Identification of Poultry Isolates. The identification of 20 isolates from spoiled broiler carcasses used in the study is shown in Table 1. Four different species of Pseudomonas comprised the pigmented group, while all nine nonpigmented isolates were identified as strains of P. fragi. The species nomencla-
TABLE 1.—Species identification1 of
pseudomonads from spoiled broiler carcasses Organism P. fragi (nonpigmented) P. boreopulis (pigmented) P. fairmontensis (pigmented) P. syncyanea (pigmented) P. convexa (pigmented) 'Based on Breed et al. (1957). 2 Out of 20.
Number isolated2 9 5 3 2 1
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 10, 2015
autoclaved); (4) sterile breast meat (one part broiler breast meat and two parts distilled water, blended in a Waring blendor and autoclaved); (5) unsterile breast meat (one part broiler breast meat and two parts distilled water, blended in a Waring blendor, but not autoclaved); (6) sterile "drip" (exudate or weep water collected from broilers bagged in polyethylene bags for 24 hrs. at 1 ° C ; after which the exudate was autoclaved); (7) sterile poultry bouillon broth—3 g. of chicken bouillon cubes (Armour), 5 g. peptone (Difco), 1000 ml. distilled water, heated to boiling to dissolve and then autoclaved; (8) sterile poultry nutrient broth (3 g. broiler breast meat, 5 g. peptone (Difco), 1000 ml. distilled water, heated to boiling to dissolve and then autoclaved).
2004
Cox, JUVEN, THOMSON, MERCURI AND CHEW
Odor Evaluation. A summary of the statistical analysis of the data colllected in our study is shown in Table 2. There were no
TABLE 2.—Statistical significance of mean odor scores obtained from analysis of variance Source of variation
Degrees of freedom Significance 3 n.s.
Media Temperature of incubation 1 n.s. Pigmented vs. nonpigmented' 1 ***2 Among pigmented 10 n.s. Among nonpigmented 8 n.s. Judges 1 n.s. 'Mean odor score of pigmented strains was 2.6; mean odor score of nonpigmented strains was 1.7. (Scores ranged from 1-4; 1 being intense off odor and 4 being identical to the uninoculated control). Significant at the 0.1% level.
significant differences in mean odor scores among media, between incubation temperatures or between judges. However, there was a highly significant difference (P < .001) in odors produced between the pigmented and nonpigmented strains of Pseudomonas, and this difference was independent of media, temperatures and judges. The nonpigmented strains produced much more intensive "off" odors in these media. The odors encountered in the study seemed to be identical to those described by Thornley et al. (1960). They reported that a putrid, ammoniacal odor appears when nonpigmented Pseudomonas reaches a level exceeding 10 7 /cm. 2 of skin. Growth Studies. To ascertain whether the differences in odor intensity were related to growth rates of the pigmented and nonpigmented strains, the growth patterns of the five different species of Pseudomonas were determined (Tables 3 and 4). Statistical analysis showed no significant differences in rate of multiplication among the five different species of Pseudomonas in either poultry bouillion broth or p-nutrient broth. Thus, the attainment of sufficient cell numbers per se, could not explain the differences in spoilage odor. Rey et al. (1969) found that proteinase activity of Pseudomonas isolated from chicken continued to increase even after cell counts reached their maximum. This may help explain the "ammoniacal" odors that were observed regardless of growth patterns. Our results suggest that the intensity of odor development during spoilage of poultry meat by Pseudomonas is more closely associated with the nature of the metabolism of the particular organism, than with differences in rates of multiplication among species or with various components of the poultry carcass. For example, there was no significant difference in odor intensity between medium no. 2 (chicken skin and water) and the other media tested that did not contain a great deal of chicken fat.
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 10, 2015
ture is based on the best classification criteria available at the time the study was conducted. However, with the recent publication of Bergey's Manual of Determinative Bacteriology (Buchanan and Gibbons, 1974), the following should be noted: P. boreopulis, P. fragi and P. syncyanea are still recognized as species, P. convexa is now called P. putida, and P. fairmontensis is not included in the new edition under any name and would now simply be classified as Pseudomonas sp. Nonpigmented strains of Pseudomonas have frequently been isolated from spoiled carcasses and in many instances were the predominant microorganisms on poultry spoiling at about 1° C. (Barnes and Shrimpton, 1958; Ingram and Thornley, 1959; Thornley et al., 1960; Barnes and Melton, 1971). Ayres et al. (1950) and Cox et al. (1974) isolated P. fragi along with other nonpigmented strains from spoiled broiler carcasses. Characteristics of the P. fragi isolates in our study were the same as those reported by these earlier investigators. Castell et al. (1959) identified P. fragi as the organism responsible for odors that develop during spoilage in fish muscle.
PSEVDOMONAS SPOILAGE IN POULTRY
2005
TABLE 3.—Growth of the different species of Pseudomonas in poultry bouillon broth at 20° C.
P. fairmontensis 2.94 3.04 2.53 3.69 4.11 4.54 5.08 7.85 9.18
Cultures P. syncyanea 2.79 3.08 2.84 3.65 3.61 4.49 5.23 7.77 9.30
P. boreopulis 2.97 3.15 3.36 3.89 4.23 4.90 5.53 8.11 9.30
P. convexa 2.77 2.69 3.23 3.62 4.08 4.15 4.98 7.57 8.98
'Logarithmic value of number of organisms per ml. of broth.
TABLE 4.—Growth of the different species of Pseudomonas in p-nutrient broth at 20° C. Cultures P. P. P. Time (hr.) fragi fairmontensis syncyanea 0 2.94' 2.73 2.72 2 3.00 2.83 2.67 4 3.11 2.95 2.65 6 3.43 3.36 3.18 8 3.43 3.18 3.84 10 3.73 3.86 4.00 13 4.54 4.08 4.08 25 7.90 7.08 7.60 73 9.52 9.40 9.38 'Logarithmic value of number of organisms per ml. of broth.
T h e s e results provide further evidence that nonpigmented strains of Pseudomonas a r e t h e principal microorganisms responsible for spoilage of poultry carcasses at refrigeration temperatures. ACKNOWLEDGEMENTS T h e authors are indebted to Mr. R. L . Richardson, Mr. R. H . D e a n , M s . Elizabeth Gable a n d Mr. Winston B o w e n for their technical assistance. Mention of specific b r a n d n a m e s does not imply e n d o r s e m e n t by the authors or institutions at which they are employed t o t h e exclusion of others not mentioned. REFERENCES Marie, A. M., and P. H. H. Gray, 1947. Aerobic bacteria that decompose cellulose, isolated from
P. boreopulis 2.97 2.89 3.20 3.58 3.83 4.28 4.74 7.65 9.43
P. convexa 2.65 2.76 2.93 3.32 3.89 3.90 4.48 8.26 9.49
Quebec soils. I. Isolation and description of the species. Can. J. Res. C, 25(6): 228-241. Ayres, J. C , W. S. Ogilvy and G. F. Stewart, 1950. Post-mortem changes in stored meats. 1. Microorganisms associated with development of slime on eviscerated cut-up poultry. Food Technol. 4: 199205. Barnes, E. M., 1960. Bacteriological problems in broiler preparation and storage. R. Soc. Health. J. 80: 145-148. Barnes, E. M., and C. S. Impey, 1968. Psychrophilic storage bacteria of poultry. J. Appl. Bact. 31: 97-107. Barnes, E. M., and W. Melton, 1971. Extracellular enzymic activity of poultry spoilage bacteria. J. Appl. Bact. 34(3): 599-609. Barnes, E. M., andD. H. Shrimpton, 1958. The effect of the tetracycline compounds on the storage life and microbiology of chilled eviscerated poultry. J. Appl. Bact. 21: 313-329. Barnes, E. M., andM. J. Thornley, 1966. The spoilage flora of eviscerated chickens stored at different temperatures. J. Fd. Technol. 1: 113-119. Breed, R. S., E. G. D. Murray and N. R. Smith,
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 10, 2015
Time (hr.) 0 2 4 6 8 10 13 25 73
P. fragi 3.00' 3.04 2.94 3.99 4.61 4.86 5.68 8.62 9.28
2006
Cox, JUVEN, THOMSON, MERCURI AND CHEW
investigation of bacterial action at chill temperatures. Sci. Agr. 15: 765-770. Nagel, C. W., K. L. Simpson, H. Ng, R. H. Vaughn and G. F. Stewart, 1960. Microorganisms associated with spoilage of refrigerated poultry. Food Technol. 14: 21-23. Rey, C. R., A. A. Kraft, R. G. Seals and E. W. Bird, 1969. Influence of temperature on some biochemical characteristics of Pseudomonas associated with spoilage of chicken. J. Food Sci. 34: 279-283. Rhodes, M. E., 1958. The cytology of Pseudomonas spp. as revealed by a silver-plate staining method. J. Gen. Microbiol. 18: 639-648. Thornley, M. J., 1960. The differentiation of Pseudomonas from the gram negative bacteria on the basis of arginine metabolism. J. Appl. Bact. 23: 37-52. Thornely, M. J., M. Ingram and E. M. Barnes, 1960. The effects of antibiotics and irradiation on the Pseudomonas-Achromobacter flora of chilled poultry. J. Appl. Bact. 23(3): 487-498. Walker, H. W., and J. C. Ayres, 1956. Incidence and kinds of microorganisms associated with commercially dressed poultry. Appl. Microbiol. 4: 345-349.
NEWS AND NOTES (Continued from page 1997) The Merck Company Foundation is funded by Merck & Co., Inc. to help provide financial support to many programs concerned with the needs of education, social welfare, health, medicine, science and civic betterment. Grants in 1974 were almost $2 million bringing total Foundation contributions since 1957 to nearly $19 million. COLORADO NOTES "Vitamin Deficiencies in Chickens and Turkeys," a 23 minute, 16 mm. color film for people needing information on the various symptoms caused by deficiencies of vitamin A, D, E, K, riboflavin, biotin, and folacin, is available from Colorado State University. Produced by Colorado State in association with Hoffmann-La Roche, Inc. of Nutley, New Jersey, the film is in seven distinct sections that can be tailored for showing by individual lecturers. Rental of the film is $30.00 per day, and it may be purchased for $250. Contact: Office of Educational
Media, Colorado State University, Fort Collins, Colorado 80523. Gordon E. Lacy, owner-operator, Highland Egg Ranch, Ault, Colorado, and Greeley Poultry, Greeley, Colorado, received the Distinguished Service Award Plaque at the recent Avian Science Association banquet at Colorado State University. At the fourth annual banquet of the Avian Science Club, Milton Landers received the Avian Science Club Scholarship of $250 and the $500 Gordon Lacy Scholarship. David Sharkey received the $300 Robbins Incubator Award for outstanding work in the area of embryology and hatchability, and the Jake Kellogg Memorial Scholarship, donated by Gordon Lacy, for $500. The Outstanding Graduating Avian Science Major Award was awarded jointly to Dianne Fagerberg and Jim Ranson. Dr. Carey Quarles, Professor of Avian Science and Assistant Department Head of Animal Sciences at Colorado State, was presented with a plaque recognizing his many contributions to the Club. Mr. Gordon Lacy, a Colorado egg producer and processor, was
(Continued on page 2018)
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 10, 2015
1957. Bergey's Manual of Determinative Bacteriology, 7th ed. The Williams and Wilkins Co., Baltimore, Md. Buchanan, R. E., and N. E. Gibbons, 1974. Bergey's Manual of Determinative Bacteriology, 8th ed. The Williams and Wilkins Co., Baltimore, Md. Castell, C. H., M. F. Greenough and J. Dale, 1959. The action of Pseudomonas on fish muscle. III. Identification of organisms producing fruity and oniony odors. J. Fish. Res. Bd. Can. 16: 13-19. Cox, N. A., A. J. Mercuri, B. J. Juven, J. E. Thomson and V. Chew, 1974. Evaluation of succinic acid and heat to improve the microbiological quality of poultry meat. J. Food Sci. 39: 985-987. Hugh, R., and E. Leifson, 1953. The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram-negative bacteria. J. Bact. 66: 24-26. Ingram, M., and M. J. Thornley, 1959. Changes in spoilage pattern of chicken meat as a result of irradiation. Int. J. Appl. Rad. Isotopes 6: 122-128. Lochhead, A. G., and G. B. Landerkin, 1935. Bacteriological studies of dressed poultry. I. Preliminary