J. Appl. Poult. Res.
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J APPL POULT RES 2007. 16:387-391
© 2007 Poultry Science Association
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Research Reports

Bacterial Populations of Broiler Carcasses Washed in Mixtures of Potassium Hydroxide and Lauric Acid1

A. Hinton, Jr.2, J. K. Northcutt, J. A. Cason, D. P. Smith and K. D. Ingram

Poultry Processing Unit, Agricultural Research Service, USDA, Russell Research Center, Athens, GA 30605

Correspondence: 2 Corresponding author: ahinton{at}saa.ars.usda.gov


   SUMMARY
TOP
 SUMMARY
DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
The bactericidal effect of mixtures of KOH and lauric acid (LA) on the microflora of broiler carcasses was examined. Carcasses were washed by shaking in KOH-LA for 1 min on a mechanical shaker. In one set of experiments, the population of bacteria recovered from carcasses following each of 3 successive washes in 1.0% KOH-2.0% LA or in distilled water (control) was enumerated. The number of total plate count bacteria, Campylobacter, and Escherichia coli in aliquots of whole-carcass-rinses of the washed carcasses was determined. Results indicated that fewer bacteria were generally recovered from carcasses after each successive wash in KOH-LA or distilled water, but significantly fewer bacteria were recovered from carcasses washed in KOH-LA than from carcasses washed in distilled water. Bacteria recovered from carcasses after the first and third wash in KOH-LA were identified using the MIDI Sherlock Microbial Identification System. Gram-positive and gram-negative bacteria were identified in the bacterial flora of carcasses washed once in KOH-LA; however, only gram-positive cocci were identified in the bacterial flora of carcasses washed 3 times in KOH-LA. Additional experiments were performed to compare the number of bacteria recovered from carcasses washed 2 times in 0.25% KOH-0.5% LA, 0.5% KOH-1.0% LA, or 1.0% KOH-2.0% LA or in distilled water (control). Results indicated that significantly fewer bacteria were recovered from carcasses washed in higher concentrations of KOH-LA than from carcasses washed in lower concentrations of KOH-LA. Findings from these experiments show that washing carcasses in KOH-LA can reduce carcass contamination by bacteria responsible for human foodborne diseases and spoilage of fresh poultry.

Key Words: potassium hydroxide • lauric acid • antimicrobial • broiler • carcass


   DESCRIPTION OF PROBLEM
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
The bacterial flora of processed poultry carcasses consists of foodborne pathogens, bacterial indicators of fecal contamination, and spoilage microorganisms [1]. Campylobacter is one of the major bacterial pathogens associated with processed poultry, and the poultry products contaminated by this bacterium are widely recognized as significant causes of foodborne outbreaks [2]. The presence of high numbers of generic Escherichia coli in processing operations may indicate high levels of contamination with fecal material that can harbor human foodborne pathogens [3]. Other bacteria found on processed poultry can produce significant economic losses by causing spoilage during refrigerated storage of fresh poultry products [4].

Chemical sanitizers, such as chlorine [5] and trisodium phosphate (TSP) [6], are currently used by commercial poultry processors to decrease microbial contamination associated with processing operations. Chlorine is widely used as a sanitizer because it is inexpensive, and it kills a wide range of microorganisms found in processing facilities [7]. Recent research has indicated that alkali salts of fatty acids (soaps) are also microbicidal toward several microorganisms found on processed broiler carcasses [8]. Mixtures of KOH and oleic, myristic, or lauric acids have been shown to kill bacteria and yeasts in vitro [9] and on poultry skin [10]. Salts of fatty acids are also used as preservatives to extend the shelf life of foods. The purpose of the present study was to examine changes in the bacterial flora of whole broiler carcasses washed in solutions of KOH and lauric acid (LA).


   MATERIALS AND METHODS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Broiler Carcasses and KOH-LA Mixtures
Broiler carcasses were obtained from a local commercial poultry processing facility. De-feathered, eviscerated carcasses were removed from the processing line and put in separate plastic bags. Bagged carcasses were placed on crushed ice and immediately transported to the laboratory for experimental trials. Upon arrival at the laboratory, necks were removed from carcasses, and carcasses were put in new plastic bags.

Freshly prepared solutions of KOH [11] and LA [12] were made on the day of the experiment. Mixtures containing 1.0% KOH and 2.0% LA (wt/vol) were prepared by dissolving KOH in distilled water, then dissolving LA in KOH solutions. Mixtures were filter sterilized by passage through 0.2-µm filters [13]. When necessary, KOH-LA mixtures were diluted with sterile distilled water to prepare less concentrated solutions.

Washing Carcasses in KOH-LA and the Enumeration and Identification of Bacteria Isolates
Four hundred milliliters of 1.0% KOH-2.0% LA or sterile distilled water (control) was added to bags containing broiler carcasses. Closed bags were placed in an automated mechanical shaker [14], and 3 carcasses were washed in distilled water or KOH-LA by shaking in the solutions for 1 min. Washed carcasses were removed from bags, and excess liquids were allowed to drip from the carcasses for 5 min. Carcasses were then placed in new plastic bags, and whole-carcass rinses (WCR) were performed to recover bacteria on the carcasses by rinsing in 400 mL of Butterfield’s phosphate buffer [15] for 1 min on the mechanical shaker. The pH of the rinsates ranged from 7.2 to 7.6. Aliquots of the buffer rinsate were removed for microbial analysis. Each carcass was washed 3 times in KOH-LA or distilled water, and each wash was followed by WCR in Butterfield’s phosphate buffer.

Serial dilutions of WCR samples were prepared in Butterfield’s phosphate buffer, and total plate count (TPC) bacteria, Campylobacter, and Escherichia coli in the rinsates were enumerated. The TPC bacteria were enumerated on Difco Plate Count Agar [16] incubated aerobically at 35°C for 48 h. Escherichia coli were enumerated on Petrifilm [17] or CHROMagar ECC [18] incubated at 35 to 37°C for 24 h. Campylobacter were enumerated on Oxoid Blood Agar Base [19] supplemented with 7.0% lysed horse blood [20] and Oxoid Campylobacter Selective Supplement (Blaser-Wang) [19]. Campylobacter plates were incubated at 42°C for 48 h in a BBL GasPak Jar System [16] with an activated BBL CampyPak Plus gas generator envelope. Morphologically distinct colonies of bacterial isolates taken from Difco Plate Count Agar inoculated with rinsates from carcasses washed 1 or 3 times in 1.00% KOH-2.00% LA were selected for identification with the MIDI Microbial Identification System [21].

Additional experiments were performed to examine changes in the population of the bacterial flora of carcasses washed in different concentrations of KOH-LA. Three carcasses were washed 2 times in 400 mL of 0.25% KOH-0.5% LA, 0.50% KOH-1.00% LA, 1.00% KOH- 2.00% LA, or sterile distilled water (control). The WCR were performed in Butterfield’s phosphate buffer as described above, and bacteria in the final rinsates were enumerated. The experiment was performed 2 times.

Statistical Analysis of Data
The number of colony-forming-units (cfu) recovered/mL of rinsate was transformed to log10 cfu/mL before conducting statistical analysis using GraphPad InStat version 4.00 for Windows 95 [22]. One-way ANOVA with Tukey-Kramer multiple comparison tests was performed to determine significant differences in group means. Unpaired t-tests were used to determine significant differences when only 2 experimental groups were compared. The P-value for all statistical tests was ≤0.05.


   RESULTS AND DISCUSSION
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Bacteria Recovered from Carcasses after Multiple Carcass Washings in KOH-LA
Washing broiler carcasses in water or KOH-LA decreased the bacterial population of the carcasses (Table 1Go). Significantly fewer TPC bacteria were recovered from carcasses washed 3 times in water than from carcasses washed only once in water and no E. coli were recovered from carcasses washed 3 times in water. Earlier research has reported that washing carcasses 10 times in 100 mL of 0.1% peptone water gradually reduced the population of aerobic bacteria and Enterobacteriaceae recovered from carcasses, although bacteria were still recovered from carcasses after 40 consecutive washes [23]. Bacteria that are loosely attached to carcasses inhabit a water film on the surface of the skin of carcasses, and these bacteria can be easily removed by the physical force of nonsurfactants that possess no bactericidal activity [24]. However, other procedures are required to remove bacteria that are more strongly attached to carcass tissues. Repeated washing of carcasses in water did not significantly reduce the number of Campylobacter recovered. The use of sanitizers, surfactants, or both is required to reduce the population of Campylobacter and other bacteria that can adhere to the surface of the skin.


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  		Table 1. Average1 log10 cfu of total plate count (TPC) bacteria, Campylobacter species, and Escherichia coli recovered per milliliter of whole-carcass rinse from broiler carcasses after multiple washing in distilled water or solution of 1.00% KOH and 2.00% lauric acid (LA)
 
Fewer bacteria were recovered from carcasses washed in 1.00% KOH-2.00% LA than from carcasses washed in distilled water (Table 1Go). Fewer TPC bacteria were recovered from carcasses washed once in KOH-LA than from carcasses washed 3 times in distilled water, although repeated washing in KOH-LA did not produce further reductions in the number of TPC bacteria recovered. Also, no E. coli or Campylobacter were recovered from carcasses after the first wash in KOH-LA. Alkali salts of fatty acids (soaps) are surfactants that improve cleansing by reducing water tension [25]; therefore, one factor that may have contributed to the ability of KOH-LA to reduce microbial contamination of carcasses could be attributed to the ability of these solutions to assist in physically removing bacteria from the surface of the skin. Furthermore, alkali salts of fatty acids are microbicides that can kill microorganisms by rupturing cellular membranes and causing leakage of the cellular contents [26]. Earlier studies have shown that Campylobacter and E. coli are susceptible to the antibacterial activity of KOH-LA in vitro [10] and on skin [8, 9]. The failure of repeated washings in KOH-LA to produce further reductions in the number of TPC bacteria recovered may indicate that the carcass microflora also contained firmly attached bacteria that were resistant to the antibacterial activity of KOH-LA. Gram-positive cocci are reported to be highly resistant to the bactericidal activity of alkaline salts of fatty acids [9, 10], and animal skin is one of their normal habitats. Although gram-negative bacteria (Acinetobacter and Shigella) were among the bacteria recovered from carcasses washed once in KOH-LA, only gram-positive cocci (Enterococcus and Staphylococcus) were recovered from carcasses washed 3 times in these solutions (Table 2Go). A combination of the surfactant activity and bactericidal activity of the KOH-LA is probably responsible for reducing the size of the bacterial population of the carcasses washed in these mixtures.


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  		Table 2. Bacterial isolates recovered from carcasses following 3 successive washings in 1.00% KOH and 2.00% lauric acid (LA)
 
Bacteria Recovered from Carcasses Washed in Different Concentrations of KOH-LA
The size of the bacterial population recovered from carcasses washed twice in different KOH-LA solutions was related to the concentration of the KOH-LA mixtures (Table 3Go). Significantly fewer TPC bacteria were recovered from carcasses washed in 0.25% KOH-0.50% LA, 0.50% KOH-1.00% LA, or 1.00% KOH-2.00% LA than from carcasses washed in water. Although Campylobacter were recovered from carcasses washed in water or 0.25% KOH-0.50% LA, none of these bacteria were recovered from carcasses washed in 0.50% KOH-1.00% LA or 1.00% KOH-2.00% LA. No E. coli were recovered from carcasses washed in 1.00% KOH-2.00% LA, but the bacterium was recovered from carcasses washed in water and in more dilute KOH-LA mixtures.


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  		Table 3. Average1 log10 of total plate count bacteria, Campylobacter species and Escherichia coli recovered per milliliter of rinsate after washing 2 times in distilled water or various concentrations of solutions of KOH and lauric acid (LA)
 
The potassium and sodium salts of the fatty acids are effective microbicides, and they are generally-recognized-as-safe substances that possess little or no human toxicity [27]. These substances also have a long history of use in food products. The ability of KOH-LA to reduce the microbial contamination of poultry carcasses indicates that salts of fatty acids deserve further study as possible sanitizers in poultry processing operations.


   CONCLUSIONS AND APPLICATIONS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
REFERENCES AND NOTES
 

  1. KOH-LA solutions are bactericidal surfactants that can reduce populations of bacteria associated with broiler carcasses.
  2. Fewer total plate count bacteria, Campylobacter spp., and E. coli were recovered from carcasses washed in solutions containing 1.00% KOH and 2.00% LA than from carcasses washed in distilled water.
  3. Gram-negative bacteria on broiler carcasses are highly susceptible to the antibacterial activity of KOH-LA, whereas gram-positive cocci on the carcasses exhibit the highest degree of resistance to the bactericidal activity of KOH-LA.
  4. Diluting the concentration of KOH-LA from 1.00% KOH-2.00% LA to 0.25% KOH-0.50% LA decreased the antibacterial activity of the solution.


   ACKNOWLEDGMENTS
 
We acknowledge the technical assistance of Jerrie Barnett, Nicole Bartenfield, Fredda G. Murray, Kathy Orr, Roger Huezo, and Jackie Hannah.


   FOOTNOTES
 
1 Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable. Back


   REFERENCES AND NOTES
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 

  1. Barnes, E. M. 1972. Food poisoning and spoilage bacteria in poultry processing. Vet. Rec. 90:720–722.[ISI][Medline]
  2. Bryan, F. L., and M. P. Doyle. 1995. Health risks and consequences of Salmonella and Campylobacter jejuni in raw poultry. J. Food Prot. 58:326–344.[ISI]
  3. Grau, F. H. 1986. Microbial ecology of meat and poultry. Pages 1–47 in Advances in meat research. 2. Meat and poultry microbiology. A. M. Pearson, T. R. Dutson, ed. AVI Publ. Co. Inc., Westport, CT.
  4. Hinton, A., Jr., J. A. Cason, and K. D. Ingram. 2004. Tracking spoilage bacteria in commercial poultry processing and refrigerated storage of poultry carcasses. Int. J. Food Microbiol. 91:155–165.[ISI][Medline]
  5. Lillard, H. S. 1979. Levels of chlorine and chlorine dioxide of equivalent bactericidal effect in poultry processing water. J. Food Sci. 44:1594–1597.[ISI]
  6. Lillard, H. S. 1994. Effect of trisodium phosphate on salmonellae attached to chicken skin. J. Food Prot. 57:465–469.[ISI]
  7. Tsai, L. S., J. E. Schade, and B. T. Molyneux. 1992. Chlorination of poultry chiller water: Chlorine demand and disinfection efficacy. Poult. Sci. 71:188–196.[ISI]
  8. Hinton, A., Jr., and K. D. Ingram. 2000. Use of oleic acid to reduce the population of the bacterial flora of poultry skin. J. Food Prot. 63:1282–1286.[ISI][Medline]
  9. Hinton, A., Jr., and K. D. Ingram. 2003. Bactericidal activity of tripotassium phosphate and potassium oleate on native flora of poultry skin. Food Microbiol. 20:405–410.[ISI]
  10. Hinton, A., Jr., and K. D. Ingram. 2005. Microbicidal activity of tripotassium phosphate and fatty acids towards spoilage and pathogenic bacteria associated with poultry. J. Food Prot. 68:1462–1466.[ISI][Medline]
  11. Spectrum Chemical Manufacturing Corp., Gardena, CA.
  12. Sigma Chemical Co., St. Louis, MO.
  13. Nalge Nunc Inc., Rochester, NY.
  14. Dickens, J. A., N. A. Cox, J. S. Bailey, and J. E. Thomson. 1985. Automated microbiological sampling of broiler carcasses. Poult. Sci. 64:1116–1120.[ISI]
  15. Food and Drug Administration/Center for Food Safety & Applied Nutrition. 2001. Bacteriological Analytical Manual. Washington, DC.
  16. Becton Dickinson and Co., Sparks, MD.
  17. 3M Microbiology, St. Paul, MN.
  18. CHROMagar ECC. DRG International Inc., Mountainside, NJ.
  19. Oxoid Limited, Basingstoke, Hampshire, UK.
  20. Lampire Biological Laboratories, Pipersville, PA.
  21. MIDI Inc., Newark, DE.
  22. GraphPad Software, San Diego, CA.
  23. Lillard, H. S. 1988. Comparison of sampling methods and implications for bacterial decontamination of poultry carcasses by rinsing. J. Food Prot. 51:405–408.[ISI]
  24. Notermans, S., and E. H. Kampelmacher. 1975. Heat destruction of some bacterial strains attached to broiler skin. Br. Poult. Sci. 16:351–361.[ISI][Medline]
  25. Kabara, J. J., R. Vrable, and S. S. F. Lie Ken Jie. 1977. Antimicrobial lipids: Natural and synthetic fatty acids and monoglycerides. Lipids 12:753–759.[ISI][Medline]
  26. Greenway, L. L. A., and K. G. H. Dyke. 1979. Mechanism of the inhibitory action of linoleic acid on the growth of Staphylococcus aureus. J. Gen. Microbiol. 115:233–245.[Medline]
  27. Kabara, J. J. 1979. Toxicological, bacteriocidal and fungicidal properties of fatty acids and some derivatives. J. Am. Oil Chem. Soc. 56:760A–767A.




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