Use of Oleic Acid To Reduce the Population of the Bacterial Flora of Poultry Skin

Transcription

1 1282 Journal of Food Protection, Vol. 63, No. 9, 2000, Pages Research Note Use of Oleic Acid To Reduce the Population of the Bacterial Flora of Poultry Skin ARTHUR HINTON, JR.* AND KIMBERLY D. INGRAM Poultry Processing and Meat Quality Unit, Agricultural Research Service, U.S. Department of Agriculture, 950 College Station Road, Russell Research Center, Athens, Georgia 30604, USA MS 00-8: Received 11 January 2000/Accepted 25 March 2000 ABSTRACT The effect of oleic acid on native bacterial flora of poultry skin was examined. Skin from commercial broiler carcasses was washed once or twice in solutions of 0, 2, 4, 6, 8, or 10% (wt/vol) oleic acid and rinsed in peptone water. Aerobic bacteria, Enterobacteriaceae, Campylobacter, and enterococci in the rinsates were enumerated. Significantly fewer aerobic bacteria, Enterobacteriaceae, Campylobacter, and enterococci were recovered from rinsates of skin washed in oleic acid than from control samples. Additionally, fewer bacteria were recovered from rinsates of skin washed in higher concentrations of oleic acid than from skin washed in lower concentrations of the fatty acid. In most cases, there was no significant difference in the number of bacteria recovered from rinsates of skin washed once or twice in solutions of oleic acid. Washing skin samples twice in 10% solutions of oleic acid significantly reduced the number of aerobic bacteria, Enterobacteriaceae, Campylobacter, and enterococci that remained attached to the skin. Campylobacter sp., Enterococcus faecalis, and Listeria monocytogenes isolates possessed the least resistance to the antibacterial activity of oleic acid in vitro, while Escherichia coli and Pseudomonas aeruginosa showed higher resistance. Enterobacter cloacae, Staphylococcus lentus, and Salmonella Typhimurium had the greatest resistance to the antibacterial activity of oleic acid. Findings indicate that oleic acid reduces the number of bacteria on the skin of processed broilers and that the fatty acid is bactericidal to several spoilage and pathogenic bacteria associated with poultry. The bacterial flora of processed poultry carcasses may include Enterobacteriaceae (12), Campylobacter (4), staphylococci, pseudomonads, and other aerobic and anaerobic bacteria (2). These microorganisms usually attach to the skin of live broilers on poultry farms (10), and a portion of these bacteria remain attached to the skin of the carcasses throughout the scalding, picking, and chilling operations in processing plants (12). Some of the bacteria on the skin of processed broiler carcasses only cause spoilage, but others are foodborne pathogens capable of causing illness or death in humans (4). Several chemicals have been proposed as sanitizers for decreasing the level of microbial contamination of processed poultry carcasses and poultry processing water. Chlorine is widely used to reduce bacterial contamination of the carcasses, to control cross contamination between carcasses, and to decrease the level of bacterial contamination of processing water in chillers in poultry processing plants in the United States (11). Additionally, studies have been conducted on the use of trisodium phosphate (6) and organic acids (5, 13) as sprays or dips to reduce the number of foodborne pathogens and spoilage bacteria on processed poultry meat. Although each of these chemical treatments reduces the bacterial population of processed carcasses, * Author for correspondence. Tel: ; Fax: ; ahinton@ars.usda.gov. contaminated poultry products continue to be a major source of human foodborne diseases (4). Fatty acids are naturally occurring, antimicrobial agents that possess little or no human toxicity (1, 7, 8). Fatty acids and their esters have been used as preservatives in some foods (8), and these substances can inhibit the growth of some bacterial pathogens (3, 15) and spoilage microorganisms (16). Additionally, salts of fatty acids (soaps) also have a long history of safe use as cleaning and disinfecting agents (7, 9). The purpose of the present study was to examine the effect of the potassium salt of the 18- carbon fatty acid, oleic acid, on the native bacterial flora of poultry skin. MATERIALS AND METHODS Skin samples and oleic acid solutions. Skin samples were taken from broiler carcasses obtained from the processing line at a local commercial poultry processing plant. Skin was removed from the carcasses, cut into 50-g pieces, placed in sterile stomacher bags (Seward Limited, London, UK), and stored at 4 C until ready for use. Skin samples were used within 5 days after being removed from the carcasses. Oleic acid solutions were made from the potassium salt of oleic acid that was a 40% (wt/vol) paste in water (Aldrich Chemical Company, Inc., Milwaukee, Wis.). Ten percent (wt/vol) solutions of oleic acid were prepared by dissolving the oleic acid paste in distilled water. The oleic acid solution was filter sterilized by passage through a 0.2- m filter (Nalge Nunc International, Rochester, N.Y.) and diluted with sterile distilled water to prepare

2 J. Food Prot., Vol. 63, No. 9 REDUCTION OF POULTRY BACTERIA WITH OLEIC ACID 1283 TABLE 1. Log CFU/ml of bacteria recovered from rinsates of poultry skin after one or after two consecutive washes in various concentrations of oleic acid Log CFU/ml a Percent oleic Enterococci Campylobacter Enterobacteriaceae Aerobic bacteria acid concentration (wt/vol) 2.07 A B BC A B B B A B A B C C C A AB AB AB AB B A AB B B B B A A A B B B A AB BC BC C BC a Values are averages standard deviation; n 6., none recovered. Within columns, different letters indicate significant (P 0.05) differences. 2, 4, 6, and 8% oleic acid solutions. All solutions were used on the same day that they were prepared. Treatments of skin samples. The effect of oleic acid on the bacterial flora of poultry skin was determined by washing the skin in the various concentrations of oleic acid. One hundred milliliters of each oleic acid solution was added to separate stomacher bags that contained 50-g skin samples, and 100 ml of peptone water (0.1% peptone, wt/vol) (Difco Laboratories, Detroit, Mich.) was added to the bags containing the control samples. The skin was washed by blending the samples in oleic acid solutions or peptone water for 1 min on normal speed in a Stomacher 400 (Seward Limited). After washing, the liquid was decanted from each bag, and 100 ml of sterile peptone water was added. The skin was rinsed by blending again in peptone water for 1 min on normal speed in the stomacher. Aliquots of the peptone water rinsates were transferred from each bag into separate sterile test tubes and set aside for microbiological analysis. Changes in the bacterial flora of rinsates of poultry skin after two successive washings in oleic acid were also examined. Primary washing of the 50-g skin samples in 100 ml of oleic acid solutions or peptone water were performed as described above. After the oleic acid solutions from the primary wash were discarded, 100 ml of a fresh oleic acid solution of the same concentration as the primary wash was added to the bag. Skin was washed for a second time by blending in the stomacher for 1 min on normal speed. The liquid from the secondary wash was then decanted, and 100 ml of peptone water was added to each bag. The skin was rinsed in the peptone water by blending in the stomacher for 1 min on normal speed. Aliquots of the rinsate were removed from the bag for microbiological analysis. The effects of oleic acid on bacteria that remain attached to the skin after two successive washings were also determined. Skin samples were subjected to primary and secondary washings by stomaching in 10% oleic acid or peptone water, as described above. After the secondary wash, all skin samples were rinsed by stomaching in 100 ml of peptone water. The rinsed samples were placed in separate, sterile Waring blender jars (Dynamics Corporation of America, New Hartford, Conn.) with 100 ml of peptone water and blended on high speed for 1 min. Aliquots of the blended suspension were set aside for microbial analysis. Control skin samples were not stomached in oleic acid or peptone water before being blended in the Waring blender. Microbiological analyses. An Autoplate 4000 Automated Spiral Plater (Spiral Biotech, Inc., Bethesda, Md.) was used to spread the rinsates and blended skin suspensions on bacteriological agar media in petri dishes. The standard plate count was performed using plate count (PC) agar (Difco) to enumerate obligately aerobic and facultatively aerobic bacteria; Enterobacteriaceae were enumerated on violet red bile salts with glucose (VRBG) agar (Oxoid Inc., Ogdensburg, N.Y.); enterococci were enumerated on enterococci (EC) agar (Difco); and Campylobacter were enumerated on the Campylobacter agar (CA) kit, Blaser (Difco). Inoculated PC, VRBG, and EC plates were incubated aerobically at 37 C, and inoculated CA plates were placed in BBL GasPak Jar Systems (Becton Dickinson Microbiology Systems, Cockeysville, Md.) with BBL CampyPak Plus Microaerophilic System Envelopes (Becton Dickinson Microbiology Systems) and incubated 42 C. Inoculated PC and VRBG agar were incubated for 24 h, and EC and CA plates were incubated for 48 h. After incubation, CFU were counted on each plate. Each experiment was repeated six times.

3 1284 HINTON AND INGRAM J. Food Prot., Vol. 63, No. 9 TABLE 2. Log CFU of bacteria recovered from the poultry skin after two consecutive washes in peptone water or 10% oleic acid solution Log CFU/g 1,2,3 Treatment Aerobic bacteria Enterobacteriaceae Campylobacter Enterococci Control Peptone water 10% oleic acid 4.55 A A B A A B A A A A B 0.67 a Values are averages standard deviation; n 6., none recovered. Within columns, different letters indicate significant (P 0.05) differences. Identification of isolates. Bacteria from rinsates of the poultry skin samples were isolated and identified. Fifty-gram samples of skin were placed in a stomacher bag, 100 ml of peptone water was added to the bag, and the skin was blended on normal speed for 1 min. The Automated Spiral Plater was used to plate samples of the peptone water rinsate on PC, VRBG, EC, CA, Listeria selective agar base, Oxford formulation (Oxoid) supplemented with Listeria primary selective enrichment supplement, UVMI (Oxoid), and mannitol salt (Difco) agar. The inoculated PC, VRBG, EC, and CA plates were incubated as described above, and Listeria selective agar base, Oxford formulation and mannitol salt plates were incubated aerobically at 37 C for 48 h. After incubation, colonies from each media were selected for identification. The Gram stain reaction of the isolates was determined. Gram-negative rods were identified with the BBL Enterotube II (Becton Dickinson Microbiology Systems) or the BBL Oxi/Ferm Tube II (Becton Dickinson Microbiology Systems). Gram-positive cocci and rods were identified with the BBL Minitek Numerical Identification System, gram-positive (Becton Dickinson Microbiology Systems). Campylobacter isolates from CA agar were confirmed with the Latex-CAMPY (jcl) Campylobacter Culture Confirmation Test (Integrated Diagnostics, Inc., Baltimore, Md.). The Salmonella Typhimurium strain ST-10 isolate was provided by the Food Animal Protection Research Laboratory of the Agricultural Research Service, United States Department of Agriculture in College Station, Tex. Inhibition of bacterial isolates by oleic acid. The effect of oleic acid on pure cultures of poultry skin bacterial isolates in vitro was determined. All bacterial isolates, except for Campylobacter, were grown at 37 C for 18 to 24 h in tryptic soy broth (Difco). The Campylobacter isolate was grown on CA agar at 42 C for 48 h. After incubation, 2 ml of peptone water was added to the surface of the CA plates, and sterile microbiological spreaders (Technical Service Consultants Ltd., Heywood, Lancs OL10 1DS) were used to scrape the Campylobacter growth from the agar. Cultures of the isolates grown in tryptic soy broth and the Campylobacter isolates were centrifuged at 5,000 g in a Hermle Z300 centrifuge (Labnet, Woodbridge, N.J.) for 10 min. Supernatants were discarded and pellets were suspended in 10 ml peptone water. Cultures were centrifuged again, the supernatant was discarded, and pellets were suspended in 10 ml of fresh peptone water. Each bacterial suspension, except for Campylobacter, was added to 5 to 10 ml of peptone water in separate Nephelo culture flasks (Bellco Glass, Inc., Vineland, N.J.) to produce an absorbance equivalent to CFU/ml. Campylobacter suspensions were added to the Nephelo culture flasks to produce an absorbance equivalent to CFU/ml. All other cultures were diluted in peptone water to achieve a final concentration of CFU/ml. Sterile oleic acid solutions (2, 4, 6, 8, and 10%) were prepared and filter sterilized, as described above. Ten-milliliter aliquots of the solutions were transferred to sterile test tubes. Control tubes contained 10 ml of sterile peptone water. One tenth of a milliliter of each isolate was added to separate tubes containing the different concentrations of oleic acid or peptone water to produce a final concentration of approximately 10 6 CFU/ml. The inoculated test tubes were mounted on a laboratory rotator (Glas- Col, Terre Haute, Ind.), and the cultures were mixed in the solutions for 5 min. The Automated Spiral Plater was used to spread contents of all inoculated tubes, except for the tubes inoculated with Campylobacter, onto PC agar. Contents of tubes inoculated with Campylobacter were plated onto CA using the Automated Spiral Plater. Inoculated PC agar plates were incubated at 37 C for 18 to 24 h, and inoculated CA plates were incubated at 42 C for 48 h in BBL GasPak Jar Systems with BBL CampyPak Plus Microaerophilic System Envelopes. After incubation, WinCount, version 1.2 (Spiral Biotech) was used to calculate CFU/ml of each isolate recovered from solutions of oleic acid and peptone water. Each experiment was repeated six times. Statistical analysis of data. All statistical analyses were performed with the GraphPad StatMate and GraphPad InStat version 4.00 for Windows 95 (GraphPad Software, San Diego, Calif., One-way analysis of variance with Tukey- Kramer multiple comparison tests were performed to determine significant differences in group means. The P-value for all analysis of variance tests was RESULTS AND DISCUSSION Increasing the oleic acid concentration of the solutions used to wash the poultry skin produced significant reductions in the number of aerobic bacteria, Enterobacteriaceae, enterococci, and Campylobacter recovered from rinsates of skin washed once or twice in solutions of the fatty acid (Table 1). Enterococci and Campylobacter from poultry skin were highly susceptible to the bactericidal activity of oleic acid, while the populations of aerobic bacteria and Enterobacteriaceae exhibited greater resistance. Some bacteria attach firmly to the skin of broilers and may still be found in carcass rinses after 40 consecutive 1-min rinses in 100 ml of 0.1% peptone (12). Finding of the present study indicate that some bacteria may remain and be recovered from rinsates even after two washings in a solution of oleic acid. Washing poultry skin samples in oleic acid also reduced the number of bacteria that remained attached to the poultry skin after rinsing (Table 2). There were significantly fewer aerobic bacteria, Enterobacteriaceae, Campylobacter, and enterococci recovered from skin washed in 10%

4 J. Food Prot., Vol. 63, No. 9 REDUCTION OF POULTRY BACTERIA WITH OLEIC ACID 1285 TABLE 3. Effect of concentration of oleic acid on poultry skin bacterial isolates in vitro Log CFU/ml recovered a Salmonella typhimurium Staphylococcus lentus Pseudomonas aeruginosa Listeria monocytogenes Enterococcus faecalis Escherichia coli Enterobacter cloacae Campylobacter sp. % oleic acid (wt/vol) 6.07 A A A B B B A B B B B B A B B B B A A A AB AB B B A A A A A A A a Within columns, different letters indicate significant (P 0.05) differences. n 3., none recovered. oleic acid than from the control samples and from the samples stomached in peptone water. The presence of fewer bacteria on poultry skin after stomaching in the oleic acid solutions was probably due to the ability of the potassium salt of the molecule to act as a surfactant that washed bacteria from the surface of the skin and to the bactericidal activity of the molecule that killed bacteria on the skin. Oleic acid inhibited the growth of most of the grampositive and gram-negative poultry skin isolates in vitro under conditions utilized in the present study; however, the susceptibility of the isolates to oleic acid varied (Table 3). Cultures of Campylobacter, Enterococcus faecalis, and Listeria monocytogenes were susceptible to low levels of oleic acid while cultures of Enterobacter cloacae, Staphylococcus lentus, and Salmonella Typhimurium exhibited the most resistance to the antibacterial activity of oleic. Bacteria with higher resistance to oleic acid, such as S. lentus and E. cloacae, may have been among the isolates recovered on PC agar from the rinsates of skin washed in 10% oleic acid; and isolates with a resistance similar to the Enterobacteriaceae, E. cloacae, may have been recovered on VRBG from rinsates of skin washed in 10% oleic acid. Fatty acids have been reported to be more effective in inhibiting the growth of gram-positive bacteria than gram-negative bacteria (7). The lipopolysaccharide layer of the cell wall of gram-negative bacteria may provide these bacteria with protection against the bactericidal activity of fatty acids (7, 17); however, some gram-positive bacteria are also resistant to the detrimental effects of fatty acids (16). Findings from the present study indicate that some gram-negative bacteria, such as Campylobacter, are highly sensitive to the antibacterial activity of oleic acid while some gram-positive bacteria, such as S. lentus, are highly resistant to the antibacterial activity of oleic acid. The antibacterial activity of fatty acids is probably due to the ability of these compounds to disrupt the membrane of the bacterial cell (2) and cause lysis of the cell (18), to inhibit bacterial nutrient uptake (14), or to form peroxides and other free radicals that inhibit bacteria growth (7). The free oleic acid molecule possesses relatively little antibacterial activity (16); however, the addition of the potassium molecule to some fatty acids increases the bactericidal activity of these compounds (19). Findings from the present study indicate that the potassium salt of oleic acid may inhibit the growth of several bacteria associated with poultry skin and that further studies should be conducted to determine if the antibacterial activity of the potassium salt of oleic acid can be practically applied to reduce the number of undesirable bacteria associated with poultry processing operations. ACKNOWLEDGMENTS The authors acknowledge the technical assistance of Kendra C. Bell, Narria C. Lofties, Katherine Orr, and Carol Stewart. REFERENCES 1. Ababouch, L., A. Chaibi, and F. F. Busta Inhibition of bacterial spore growth by fatty acids and their sodium salts. J. Food Prot. 55:

5 1286 HINTON AND INGRAM J. Food Prot., Vol. 63, No Barnes, E. M Food poisoning and spoilage bacteria in poultry processing. Vet. Rec. 90: Bergsson, G., J. Arnfinnsson, S. M. Karlsson, O. Steingrimsson, and H. Thormar In vitro inactivation of Chlamydia trachomatis by fatty acids and monoglycerides. Antimicrob. Agents Chemother. 42: Bryan, F. L., and M. P. Doyle Health risks and consequences of Salmonella and Campylobacter jejuni in raw poultry. J. Food Prot. 58: Cox, N. A., A. J. Mercuri, B. J. Juven, J. E. Thomson, and V. Chew Evaluation of succinic acid and heat to improve the microbiological quality of poultry meat. J. Food Sci. 39: Hwang, C., and L. R. Beuchat Efficacy of selected chemicals for killing pathogenic and spoilage microorganisms on chicken skin. J. Food Prot. 58: Kabara, J. J Toxicological, bacteriocidal and fungicidal properties of fatty acids and some derivatives. J. Am. Oil Chem. Soc. 56:760A 767A. 8. Kabara, J. J Medium-chain fatty acids and esters. In P. M. Davidson and A. L. Branen (ed.), Antimicrobials in foods. Marcel Dekker, Inc., New York. 9. Kabara, J. J., R. Vrable, and S. S. F. Lie Ken Jie Antimicrobial lipids: natural and synthetic fatty acids and monoglycerides. Lipids 12: Lahellec, C., and P. Collin Relationship between serotypes of salmonellae from hatcheries and rearing farms and those from processed poultry carcasses. Br. Poult. Sci. 26: Lillard, H. S Effect on broiler carcasses and water of treating chiller water with chlorine or chlorine dioxide. Poultry Sci. 59: Lillard, H. S Incidence and recovery of salmonellae and other bacteria from commercially processed poultry carcasses at selected pre- and post-evisceration steps. J. Food Prot. 52: Lillard, H. S., L. C. Blankenship, J. A. Dickens, S. E. Craven, and A. D. Shackelford Effect of acetic acid on the microbiological quality of scalded picked and unpicked broiler carcasses. J. Food Prot. 50: Macaskie, L. E Inhibition of growth of Brochothrix thermosphacta by palmitic acid. J. Appl. Microbiol. 52: McKellar, R. C., A. Paquet, and C.-Y. Ma Antimicrobial activity of fatty N-acylamino acids against gram-positive foodborne pathogens. Food Microbiol. 9: Ouattara, B., R. E. Simard, R. A. Holley, G. J.-P. Piette, and A. Bégin Antibacterial activity of selected fatty acids and essential oils against six meat spoilage organisms. Int. J. Food Microbiol. 37: Russell, A. D Mechanisms of bacterial resistance to non-antibiotics: food additives and food and pharmaceutical preservatives. J. Appl. Bacteriol. 71: Tsuchido, T., N. Yokosuka, and M. Takano Isolation and characteristics of a Bacillus subtilis mutant tolerant to the lytic action of sucrose esters of long-chain fatty acids. J. Ferment. Bioeng. 75: Wang, L., and E. A. Johnson Inhibition of Listeria monocytogenes by fatty acids and monoglycerides. Appl. Environ. Microbiol. 58: