MOMW Journal of Food Protection, Vol. 69, No. 7, 2006, Pages 1611-16/S
Antimicrobial Activity of Potassium Hydroxide and Lauric Acid against Microorganisms Associated with Poultry Processingt ARTHUR HINTON, JR.' ANI) KIMBERLY D. INGRAM poultry Processing Unit, Agricultural Research Service, U.S. Department of Agriculture, 950 College Station Road, Russell Research Athens, Georgia 30605, USA
6 Cente,,
MS 05-623: Received 15 December 2005/Accepted 21 February 2006
ABSTRACT The antimicrobial activity of solutions of potassium hydroxide (KOFI) and mixtures of KOH and laurie acid against microorganisms associated with poultry processing was determined. In vitro tests were performed by enumerating viable microorganisms recovered from bacterial cultures suspended in peptone water (control) and in solutions of 0.1% KOH or mixtures of 0.1% KOH and 0.25 or 0.501/c laurie acid. Additional studies were conducted to identify changes in the native microbial flora of poultry skin washed in distilled water. KOH. or KOH—lauric acid. Although results of in vitro studies indicated that significantly fewer bacteria (P -^ 0.05) were recovered from cultures suspended in KOl-! than from cultures suspended in peptone water, there were also significantly fewer bacteria recovered from cultures suspended in KOH—lauric acid than from cultures suspended in KOH. Results of experiments with broiler skin indicated that although rinsates of skin washed in 1.0% KO1-I solutions contained significantly fewer total aerobic bacteria and enterococci than did skin washed in water, significantly fewer of these microorganisms were generally recovered from rinsates of skin washed in mixtures of 1.0% KOH and 0.5. 1.0. 1.5, or 2.0% laurie acid than from skin washed in KOH alone. Washing of broiler skin in solutions of 0.25 to 1.00% KOH or mixtures containing these concentrations of KOH and two parts lauric acid (wtivol) also significantly reduced the populations of bacteria and yeasts in the native flora of broiler skin. Enterococci, lactic acid bacteria, and staphylococci in the native flora of the skin had the highest level of resistance to the bactericidal activity of KOH—lauric acid. These findings indicate that the antimicrobial activity of KOH—lauric acid is significantly greater than that of KOH alone in vitro and on poultry skin. Thus. KOH—lauric acid may he useful for reducing the level of microbial contamination associated with poultry processing.
Processed poultry products may be contaminated with pathogenic microorganisms that cause human foodborne diseases and with spoilage microorganisms that reduce the shelf life of poultry products (3). Canip.vlohacter and Salmonella are currently recognized as the major bacterial pathogens associated with poultry (4, 25), but poultry products contaminated with Lisleria m000cvrogene s (7). Staphvlococcu.c aureus, and Clostridium perfringens (25) also cause a significant number of cases of human foodhorne diseases. Spoilage microorganisms such as Pseudomona,s spp., BrochotrLv therrnosphacta, and yeasts (3, ii) and fecal indicator bacteria such as Escherichia co/i (6) also may represent a significant portion of the microbial flora of processed poultry. Several chemicals are currently used as sanitizers to reduce microbial contamination in poultry processing operations (4). Chlorine is the most widely used sanitizer in commercial poultry-processing facilities because it is inexpensive and relatively effective against microorganisms found in poultry-processing environments (19. 26). The use of the alkaline sanitizer trisodium phosphate also has been *
Author for correspondence. Tel: 706-546-3621; Fax: 706-546-3633:
E-mail:
[email protected]. t Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may he suitable.
approved by the U.S. Department of Agriculture for the reduction of Salmonella contamination of poultry carcasses during commercial processing operations (9). Despite the use of these and other available sanitizers, microbial contamination of processed poultry continues to he a significant food safety issue. and research on technology to extend the shelf life of fresh poultry continues. Previous research has indicated that the antimicrobial activity of mixtures of potassium triphosphate and fatty acids can reduce populations of microorganisms in vitro and on skin of processed broiler carcasses (12-14). Fatty acids are naturally occurring microbicides that have little or no human toxicity (15). The potassium and sodium salts of fatty acids (soaps). which are formed by adding potassium hydroxide (KOH) or sodium hydroxide to fats, also possess antimicrobial activity (1). These compounds have a long history of safe use as cleansers and food preservatives (15. 16). The purpose of the present study was to examine the antimicrobial activity of KOH and mixtures of KOH and laurie acid in vitro and on poultry skin. MATERIALS AND METHODS Antimicrobial solutions. Solutions containing various concentrations of KOH (Sigma Chemical Co.. St. Louis, Mo.) or mixtures of KOl-I and laurie acid (Sigma) were prepared. KOH solutions were made by dissolving KOH pellets in distilled water (wtivol), and mixtures of KOH and laurie acid were prepared by
612 HINTON AND INGRAM
J. 100(1 Piot.. Vol. 69, NO 7
TABLE l. Microorganisms recovered q ,fter culture in peptone water, KOH solution, or KOH—laurjc acid So/ulionsa Microorganisms recovered (log CFU/ml)
-
Acinetohacier Aero,nonas Brochotrix Camp yfobacter Clostridjwn Enterobacrer Solution calcoacc',(cus sohpja the rniocpha eta jejuni pefringens cloacae Peptone water 5.63 ± 0.19c 5.73 ± 0.09c 5.73 ± 0.19c 6.35 ± 0.04B 5.93 ± 0.14B 5.22 ± 0.05 u 0.10% KOH 2.93 ± 0.06 B 3,44 ± 0-10B 3.09 ± 0.04 B 1.63 ± 0.35 A NRA 1.56 ± 0.24 A 0.10% KOH + 0.025% lauric acid NR A NR A NR A NR A NR A NR A 0.10% KOH + 0.05% laurie acid NR A NR A NR A NR A NR A NR A a Values are means ± standard deviations. n = 3. NR, none recovered. Within columns, different letters indicate significant differences (P 0.05). dissolving lauric acid in KOH solutions. All solutions were filter sterilized by passage through 0.2-p.m-pore-site filters (Nalge Nunc International, Rochester, N.Y.). The final pH of the solutions was measured with an electronic pH meter (Corning Inc., Corning, N. Y.). Microbial isolates. The antimicrobial activity of KOH and KOH—lauric acid solutions in vitro was determined by enumerating viable microorganisms recovered from microbial suspensions mixed in the solutions. Previously identified microbial isolates that had been recovered from rinsates of commercially processed broiler carcass skin (13) were used in the present study. A B. thermosphacta isolate was recovered from rinsale.s of broiler carcass skin plated on streptomycin sulfate thallous acetate actidionc agar (2) and incubated at 20°C for 3 days.. A C. perfringens isolate was recovered from skin rinsates plated on Perfringens agar base with egg emulsion and tryptose sulfite cycloserine selective supplement (Oxoid Ltd.. Basingstoke, Hampshire, UK) and incubated anaerobically in an anaerobic chamber (Coy Laboratory Products inc., Grass Lake, Mich.) at 35°C for 18 to 24 h. B. the rmosphacta and C. peifringens isolates were identified with the MIDI Sherlock Microbial Identification System, as previously described (11, 24). Fresh cultures of Acinetobacter ca/coaceticus, Aeromonas .cobr,a, B. thenno.cphacta Entero/,acier cloacae, Enterococcus faeca/is, E. co/i P01 , L. ?nonocvtoçenes Salmonella Enteritidj s, Salmonella Typh im un urn, S. aureus, Staph viococcus chromogenes KP06. and Pseudomonas aeruginosa were grown aerobically in tryptic soy broth (Difco, Becton Dickinson, Sparks. Md.) for 18 to 24 h at 35°C. Cultures of C. perfringens were grown anaerobically in reinforced clostridia medium broth (Difco, Becton Dickinson) for 18 to 24 h at 35°C. Campv/obacterjejuni cultures were grown on Remel blood agar (Remel Inc., Lenexa, Kans.) for 48 h at 42°C under microaerophilic conditions in a GasPak jar (BBL, Becton Dickinson) with an activated CampyPak Plus Hydrogen + CO2 with Integral Palladium Catal y st (Becton Dickinson). All microbial cultures were harvested and suspended in a solution of 0.1% Bacto Peptone water (Difco, Becton Dickinson) as previously described (13). In vitro antimicrobial tests. In vitro trials were conducted by adding 0.1 ml of each microbial isolate culture to separate tubes containing 10 ml of peptone water (control), 0.1% KOH solution, or mixtures of 0.1% KOH and 0.025 or 0.050% laurie acid to produce a final microbial concentration of approximately 106 CFU/ml. Test tubes containing the suspensions were placed on a laboratory rotator (Glas-Col, Terre Haute, hid.) and mixed for S mm. Aliquots of the microbial suspensions were then removed and mixed with an equal volume of Butterfield's phos-
phate-buffered dilution water (28) to neutralize the alkalinity of the KOH or KOH—lauric acid solutions. Microorganisms in the suspensions were then enumerated by plating on the appropriate agar medium with an Autoplate 4000 automated spiral plater (Spiral Biotech, Bethesda. Md.). Cultures of E. cloacae, E. fliecalis, E. coil, L. monocvlogenes, Salmonella Enteritidis, Salmonella Typhirnurium, S. aureus, S. chromogenes, and P. aerugino.s'a were plated onto plate count agar (PCA: Difco, Becton Dickinson) and incubated aerobically at 35°C for 24 to 48 h. Cultures of C. jejuni were plated on Remel blood agar and incubated rnieroaerobically at 42°C for 48 h. Cultures of C. pefringens were plated on reinforced clostridia agar (Difeo, Becton Dickinson) and incubated anaerobically at 35°C for 18 to 24 h. After incubation, the concentration of each isolate recovered from the peptone water, KOH, or KOH—lauric acid suspensions was determined. Each experiment was repeated three times. Determinatioji of the effect of KOH and KOH—laurie acid on the microbial flora of poultry skin. Broiler carcasses were taken from the processing line of a local commercial poultry processing plant immediately after feathers had been removed by a mechanical picker. These carcasses were placed on crushed ice and transported to the laboratory, where the carcass skin was manually removed. The skin was cut into 50-g pieces, placed in sterile stomacher bags (Seward Ltd., London. UK). and stored at 4°C until used. All skin samples were used within 5 da y s after being removed from the carcasses. Differences in the number of total aerohcs and enterococci recovered from broiler skin washed in mixtures containing KOH and various concentrations of laurie acid was determined. Skin samples were washed in 100 ml of sterile distilled water (control). 1.0% KOH solution, or mixtures of 1.0% KOI-1 and 0.5, 1.0, 1.5. or 2.0% laurie acid in a Stomacher 400 (Seward) on high speed for 1 mm. The washed skin was then transferred to a sterile stomacher bag containing 100 ml of fresh Butterfield's phosphate-buffered dilution water and stomached for I nun on high speed. Aliquots of the buffer rinsates were removed for immediate microbial analyses. An automated spiral plater was used to plate the rinsates on the skin samples on agar media. Rinsates were plated on PCA and incubated at 35°C for 24 It to enumerate total aerobic bacterial flora and on enterococci agar (Difco. Becton Dickinson) incubated at 35°C for 48 h to enumerate enterococci Differences in the microflora of broiler skin washed in solutions containing various concentrations of KOI-I or mixtures of KOH with two parts laurie acid (wtivol) were also determined. Skin samples were washed in distilled water (control), in solutions of 0.25. 0.50. 0.75, or 1 fific/ K011. or in nH\tUrcs oil) :; KOH
"u. 7
J FOU'- 'i-oi.. \'ol. 69.
1613 ANTIMICROBIAL ACTIVITY OF K011 ,.\r\1) I AIJRIC ACID
TABLE 1. Extended
Microorganisms recovered (log CFU/ml)
-
I'seudonionas So/inane/la Solnione//o EnterOCOcclL Escherichia Listeria aeruginoso Enterilidis Typhimurium ,nonocviogenes faeca/is - co/i P0! ± 0.19 c 5.70 ± 0.09 c 6.38 ± 0.06 c 6.59 ± 0.04 B A 5.01 ± 0.11 it 2.12 ± 0.20 B 2.48 ± 0.11 B 1.56 ± 0.24
5.66 ± 0.17 c 5.97 ± 0.04 c 6.82
.33 ± 0.61
B 3.60
± 0.09
1.39 ± 0.18 A 1.45 ± 0.28
B
A NR
NRA
NR NR NR NRA and 0.50% lauric acid. 0.50% KOH and 1.00% laurie acid, 0.75% KOH and 1.50% lauric acid, or 1.00% KOH and 2.00% laurie acid. Except for the E. co/i, selected populations of the microbial flora of the skin rinsates were enumerated by plating the rinsates on the appropriate microbial media with an automated spiral plater. Total plate counts were determined on PCA incubated at 35°C for 24 h. Campy/oboe/er counts were determined on Oxoid blood agar base supplemented with 7.0% lysed horse blood (Lampire Biological Laboratories, Pipersvillc, Pa.) and Oxoid campvlobacicr selective supplement (Blaser-Wang) incubated at 42°C for 48 h. Enterococci counts were determined on enterococci agar incubated at 35°C for 48 li. Lactic acid bacteria counts were determined on lactic acid bacteria agar (2) incubated anaerobically at 35°C for 48 h. Psychrolrophic bacteria counts were determined on PCA incubated at 4°C for It) days. Staphylococci counts were determined on Bacto Baird-Parker agar base (Difco. Becton Dickinson) supplemented with egg yolk tellurite enrichment agar (BBL. Becton Dickinson) incubated at 35°C for 48 h. Yeast counts were determined on acidified Sabouraud dextrose agar (Difco, Becton Dickinson) incubated at 28°C for 3 days. E. co/i coLints were determined on coljform/E. co/i Petrifllm (3M Co., St. Paul. Minn.) incubated at 35°C for 24 to 48 h. Morphologically distinct colonies were removed from Baird-Parker agai enterococci agar PCA, and lactic acid bacteria agar. Additional studies were conducted to confirm the identity of staphylococci, enterococci. and lactic acid bacteria that exhibited the highest resistance to the antimicrobial activity of KOH- laurie acid mixtures on broiler skin. Skin samples were washed in a mixture of 1.0% KOH and 0.5% laurie acid, and skin rinsates were plated on PCA. Baird-Parker agar, enterococci agar. and lac-
TABLE 2. Mean ph of solutions of KOhl and KOH-Iauric a ul Solution composition (4. wi/volt KOH 1,auric acid 0.10 0.0 0.25 0.0 0.50 0.0 0.75 0.0 1.00 0.0 0.10 0.025 0.10 0.05 0.25 0.5 0.50 1.00 0.75 1.50
pH
12.22 ± 0.15 AB 12.48 ± 0.23 ABC 12.83 ± 0.21 BC 13.05 ± 0.24 c 13.19 ± 0.17 c 12.15 ± 0.07 A 12.04 ± 0.15 A 12.12 ± 0.39 A 12.27 ± 0.30 An 12.48 ± 0.24 ABC
Values are means ± standard deviations. n = 3. Different letters indicate significant differences (P ::-^ 0.05).
Staphylococcus stelplivlococcus cli roinogenes aureus KPOÔ
5.22 ± 0.05 n 5.22 ± 0.05 c 1.56 ± 0.24 A 3.97 ± 0.08 B
NRA NR NRA NRA NR NRA NR
tic acid bacteria agar and incubated as described. Isolates from each medium were identified with the MIDI Sherlock Microbial Identification System. Statistical analysis. 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 (ANOVA) with Tukey-Kramer multiple comparison tests was performed to determine among group means. Differences were considered significant at P 5 0.05.
RESULTS AND DISCUSSION Results of in vitro studies indicated that solutions of KOH and solutions of KOH-lauric acid possess antimicro-
bial activity against several microor g anisms associated with processed poultry (Table I). Significantly fewer gram-negative and gram-positive bacteria were recovered from cul-
tures suspended in KOH than from cultures suspended in peptone water. However, mixtures of KOH and laurie acid were more bactericidal than were solutions of KOH alone. The only viable cells recovered from mixtures of 0.10% KOH and 0.025% laurie acid were from cultures of the gram-positive coccus E. faeca/is and the gram-negative rod E. co/i. However, no viable cells were recovered from any cultures suspended in mixtures of 0.10% KOH and 0.050% laurie acid. Although alkaline sanitizers such as tripotassium phosphate (13) and trisodium phosphate (5, 20) may significantly reduce populations of gram-negative bacteria, these sanitizers are generally more effective for killing gram-positive bacteria and yeasts (13). The mechanism of
the antimicrobial activity of alkaline sanitlzcrs is not known, but this activity may he related to the high pH of these compounds (5). The pH values of solutions of KOH and KOH-lauric acid are greater than 12.0 (Table 2), and
bactericidal solutions of 4.0% potassium triphosphate have a pH of 12.4 (14). However, fatty acids are known to kill microorganisms because of their surfactant activity and their ability to cause cellular lysis by disrupting cell membranes (10, 17, 27). The antimicrobial activity of fatty acids is generally more effective against gram-positive bacteria and yeasts (18, 29) than against gram-negative bacteria (21,
23) because the protective lipopolysaccharide layer of gram-negative bacteria serves as a barrier to penetration by fatty acids (22. 23). The ability of KOH-lauric acid solutions to kill gram-positive and grani-negative bacteria and
614
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TABLE 3. Bacteria recovered on plate count agar and enterococci agarfroin rinsates of poultry skin washed in distilled water, KOH, or KOH-/auric acida
N\
C - C - t C ri -
+1 +1 +1 +1 + +1 +1
Bacteria recovered (log CFU/mI)
M ' t
(N
Rinse solution Plate count agar Enterococci agar 0
Distilled water
4.24 ± 0.190 3.03 ± 0.44c 1.0% KOH 3.55 ± 0.14c 1.98 ± 0.41 B 1.0% KOH + 0.5% lauric acid 2.94 ± 0.29 n 1.54 ± 0.48 AB 1.0% KOFI + 1.0% lauric acid 2.29 ± 0.54 A NR A 1.0% KOH + 1.5% lauric acid 2.51 0.36 ± AB NR 1.0% KOH + 2.0% lauric acid 2.19 ± 0.33 A NR A
U
CCCCCCCCC +1 +r +1 + + +1 +i +1 + OC
cn
C
(NC
L1
( c
° Values are means ± standard deviations, n = 6. NR, none re-
-
-
r' (N c' (N
r--r'
covered. Within columns, different letters indicate significant differences (P S 0.05).
yeasts in vitro is probably due to the combined microbicidal activity of both compounds. KOM solutions and KOH-laurjc acid solutions also exhibited antimicrobial activity against the native microflora of processed broiler skin. Significantly fewer total bacteria and enterococci were recovered from rinsates of skin washed in KOH solutions than from those of skin washed in distilled water, and significantly fewer bacteria were recovered from skin washed in KOH-lauric acid than from skin washed in distilled water or KOH (Table 3). As the concentration of lauric acid in the KOH-laurjc acid solutions used to wash the skin was increased, the population of total bacteria and enterococci recovered from the skin rinsates generally decreased. Washing broiler skin in KOH or KOH-lauric acid solutions also reduced the population of several other types of microorganisms in the native microflora ofpoultry skin (Table 4). Although washing skin in KOH alone generally reduced the population of Cainpvlohacter, E. co/i, enterococci, lactic acid bacteria, pseudomonads, staphylococci, and yeasts recovered from skin rinsates, significantly fewer of these microorganisms were recovered from skin washed in solutions of KOH-laurjc acid. The gram-positive enterococci, lactic acid bacteria, and staphylococci exhibited the highest degree of resistance to the antibacterial activity of these solutions. The microbicidal activity of KOH-lauric acid may have been reduced by proteins and fats in the skin; therefore, more concentrated solutions were required to reduce microbial populations on chicken skin than in vitro. The identities of some of the gram-positive rods and cocci that were recovered from skin washed in 1.0% KOH and in 0.05% lauric acid are listed in Table 5. Fatty acids and their salts (15) are generally recognized as safe (GRAS) substances that have a long history of use in food products, and KOH also is a GRAS substance. The use of chlorine as a disinfectant during poultry processing is currently under scrutiny because toxic chiororganic compounds may be formed when chlorine comes in contact with proteins and fats during processing (8). .Additionally, there is an increasing concern about the environmental impact of the high levels of phosphate contained in wastewater from processing facilities that use trisodium phos-
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Bacterial isolates recovered on plate count agar. Baird-Parker agar, enlerococci agar, and lactic acid bacteria agar from TABLE 5. broiler chicken skin rinsed in a mixture of I,O'/r KOH and 0.57r lauric acid' Lactic acid bacteria agar Enterococci agar Baird-Parker agar Plate count agar Bacillus ,narinus MicrOcoCCUc lutei1s GC subgroup C Morare11a os/eon S. aureus GC subgroup C S. chrotflogei(' S. cohni-cohti S. saprophyticus
Lactobacillus oris Corvnehacteriu,n amvcolatum Enterococcus ai'ium L. salivarius-,salisariiLs A subgroup E. faecium GC Staph ylococcus sitnulans intestinalis Streptococcus E. faecium GC subgroup B S. xlosus
'Identification performed with the MIDI Sherlock Microbial Identification System.
phate as a sanitizer. The safety and effectiveness of sanitizers that contain fatty acids and their salts indicate that these products deserve further study to determine whether they may be used as practical alternatives to sanitiLers currently in use in poultry processing operations. ACKNOWLEDGMENTS The authors acknowledge the technical assistance of Jerrie Barnett and Fredda G. Murray.
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