Bactericidal activity of alkaline salts of fatty acids towards bacteria associated with poultry processing

Introduction

Several chemicals are currently used as sanitizers by commercial poultry processors to reduce microbial contamination of poultry processing operations (Oyarzabal, 2005). Chlorine is the most widely used sanitizer in commercial poultry processing facilities in the United States because it is inexpensive and relatively effective against microorganisms found in processing environments (Russell & Keener, 2007). Recent research has indicated that use of chlorine during processing can lead to the production of carcinogenic compounds such as trihalomethanes, however. Other poultry processing sanitizers include acidified sodium chlorite, trisodium phosphate, cetylpyridinium chloride, chlorine dioxide, and organic acids. Despite the use of these sanitizers, poultry meat contaminated by pathogenic bacteria continues to be widely cited as a significant cause of human foodborne illnesses (Friedman et al., 2004).
Fatty acids (FA) are naturally occurring microbicides that have a long history of safe use as cleansers and food preservatives (Kabara et al., 1977). These substances are components of numerous vegetable and animal oils and have little or no human toxicity. FA are also generally recognized as safe (GRAS) by regulatory agencies. Potassium or sodium salts of FA are formed when FA are dissolved in solutions of potassium hydroxide (KOH) or sodium hydroxide (NaOH), respectively. These alkaline salts of FA are surfactants that also possess antimicrobial activity. Alkaline salts of FA can inhibit the growth of several bacteria and yeasts associated with poultry processing in vitro (Hinton & Ingram, 2005), on poultry skin (Hinton & Ingram, 2003), and on whole broiler carcasses (Hinton et al., 2009). Alkaline salts of lauric acid have been examined as possible sanitizers for poultry processing, but other antimicrobial FA (Amalaradjou et al., 2006; Bergsson, et al., 2002; Hermans et al., 2010; Solis de los Santos et al., 2008) could possibly be used for this purpose.
The agar diffusion method can be used to examine the antimicrobial activity of antibiotics and other inhibitory substances (Boney et al., 2008). The assay is conducted by placing antimicrobial test agents in agar wells or on paper discs on agar plates seeded with test microorganisms. Inhibitory substances diffuse into the seeded agar during incubation, and zones of inhibition of microbial growth are produced in areas where inhibitory concentrations of the test substance are present. Therefore, the agar diffusion assay can be used to quickly screen potential antimicrobial compounds for inhibitory activity against targeted microorganisms. The purpose of the present study was to examine the ability of the agar diffusion assay to assess the antibacterial activity of formulations of alkaline salts of several FA towards bacteria associated with poultry processing.

Materials & Methods

Fresh cultures of Acinetobacter calcoaceticus, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Typhimurium, and Staphylococcus simulans were grown aerobically in Difco Tryptic Soy Broth. All cultures were incubated at 35^oC, except for L. monocytogenes which was incubated at 30^oC, for 18-24 h at 100 rpm in a Model D76 gyrotory water bath shaker. Campylobacter jejuni cultures were grown on Blood Agar (Becton Dickinson, and Co.) supplemented with 7% lysed horse blood for 48 h at 42^oC under microaerophilic conditions in a BD BBL GasPak Jar with a BD GasPak^TM EZ Campy Gas Generating Pouch System. All cultures were harvested and suspended in a solution of 0.1% Difco Bacto Peptone to produce bacterial suspensions containing 10⁷ cfu/ml.

Tempered agar media was seeded with bacterial cultures to produce an inoculated agar media containing 10⁶ cfu/ml. Bacterial suspensions were mixed in tempered agar, and 25 ml aliquots of agar were added to Petri plates to produce an agar layer approximately 6.5 mm thick.

Alkaline salts of lauric, capric, caprylic, caproic, and myristic acids were prepared by dissolving 0.5 M of each FA into separate volumes of 1 M KOH. The pH of these mixtures was adjusted to 10.5 by adding dilute citric acid solutions to the FA mixtures. All solutions were filter sterilized as described above.

A suction device (Bell & Grundy, 1968) equipped with metal tubing was used to make 8 mm wells in the solidified, seeded agar. For each assay, wells in agar plates were filled with 0.1 ml of the appropriate FA-KOH solution. All plates, except plates inoculated with C. jejuni, were then incubated aerobically at 35^oC for 18-24 h. Blood Agar plates seeded with C. jejuni were incubated microaerophilically at 42^oC for 48 h. After incubation, zones of inhibition of bacterial growth around the agar wells were measured from the outside edge of the well to the area of visible bacterial growth using Traceable^Ò Carbon Fiber Digital Calipers.

All statistical analyses were performed with the GraphPad StatMate^TM and GraphPad InStat^® version 3.05 for Windows 95 (GraphPad Software, San Diego, CA, USA). One-way Analysis of Variance (ANOVA) with Tukey-Kramer Multiple Comparison tests was performed to determine significant differences in group means. The P value for all ANOVA tests was < 0.05.

Results & Discussion

In the present study, alkaline salts of different FA varied in their ability to inhibit bacterial growth (Table 1). Caproic acid-KOH only exhibited antibacterial activity towards C. jejuni. Caprylic acid-KOH inhibited growth of 3 of the Gram negative isolates, C. jejuni, E. coli, and P. fluorescens. The size of the zones of inhibition of C. jejuni and P. fluorescens were significantly larger than the size of the zones of E. coli. Capric-KOH and lauric acid-KOH exhibited antibacterial activity towards all 8 of the bacterial isolates. The size of the zones of inhibition produced by capric acid-KOH was Salmonella Typhimurium < E. coli < A. calcoaceticus < L. monocytogenes and S. simulans < P. fluorescens < E. faecalis < C. jejuni. The size of the zones of inhibition produced by lauric acid-KOH was E. coli < A. calcoaceticus < Salmonella Typhimurium and E. faecalis < C. jejuni, S. simulans, and P. fluorescens < L. monocytogenes. Myristic acid-KOH was only inhibitory towards the Gram positive cocci, E. faecalis and S. simulans, and the size of the zones of inhibition for E. faecalis were significantly larger than the zones for S. simulans.

Various FA have been reported to possess antimicrobial activity (Kabara et al., 1977). The bactericidal activity of FA has been related to the ability of these substances to cause lysis of bacterial cells by disrupting cellular membranes. The complex nature of the lipopolysaccharide layer of Gram negative bacteria may provide protection to some of these bacteria against the antibacterial activity of FA. Among the Gram negative isolates, the fragile C. jejuni exhibited the highest degree of susceptibility to bactericidal activity of FA. Salmonella Typhimurium exhibited greater resistance to the shorter chained, capric acid- and caprylic-KOH, than E. coli which exhibited greater resistance to the longer chained lauric acid-KOH. Acinetobacter and Pseudomonas species possess similar cell walls, but Acinetobacter sp. have an additional detached membrane not found in pseudomonas (Thorney & Sleytr, 1974) which may give Acinetobacter a higher degree of resistance to FA than pseudomonas. However, the thicker cell wall of Gram positive bacteria may provide these bacteria resistance against the antibacterial activity of some FA. In general, the shorter chained FA (e.g. caprylic) were more inhibitory towards Gram negative bacteria while longer chained FA (e.g. myristic) were more inhibitory towards Gram positive bacteria.

Table 1. Size of Zones of Inhibition (mm) of Bacteria Associated with Poultry Processing by Alkaline Salts of Fatty Acids

	Fatty Acid¹
Bacterial Isolate	Caproic	Caprylic	Capric	Lauric	Myristic
*Acinetobacter calcoaceticus*	0.00^a	0.00^a	4.39^c	2.43^ab	0.00^a
*Campylobacter jejuni*	5.97^b	4.97^c	14.97^g	4.04^c	0.00^a
*Enterococcus faecalis*	0.00^a	0.00^a	11.73^f	3.97^b	7.59^c
*Escherichia coli*	0.00^a	1.79^b	2.17^b	2.17^a	0.00^a
*Listeria monocytogenes*	0.00^a	0.00^a	5.44^d	10.15^d	0.00^a
*Pseudomonas aeruginosa*	0.00^a	4.47^c	6.70^e	4.72^c	0.00^a
*Salmonella Typhimurium*	0.00^a	0.00^a	0.15^a	2.97^b	0.00^a
*Staphylococcus simulans*	0.00^a	0.00^a	5.85^d	4.62^c	0.60^b

^{1 Values are averages + standard deviation of zones of inhibition. n = 15.
A-G Within columns, different letters indicate significant (P < 0.05) differences in size of zones of Alkaline salts of these FA are also surfactants, and lauric acid-KOH mixtures have shown some potential for use as sanitizers that can reduce carcass contamination by spray washing eviscerated broiler carcasses with these mixtures (Hinton et al., 2009).}

Conclusions

Findings of this study provide additional evidence that alkaline salts of several FA are bactericidal towards several bacteria associated with poultry processing. These FA differ in their ability to inhibit bacterial growth however; therefore mixture of two or more of these acids will probably be required to formulate a sanitizer with a wide antibacterial spectrum. Furthermore, results indicate that the agar diffusion assay can be used as a rapid method for examining inhibitory activity of formulations of alkaline salts of FA.

References

Amalaradjou MAR, Annamalai TV, Marek P, Rezamand P, Schreiber D, Hoagland T, Venkitanarayanan K. 2006. Inactivation of Escherichia coli O157:H7 in cattle drinking water by sodium caprylate. J Food Protect. 69:2248-2252.

Bell SC & Grundy WE. 1968. Preparation of agar wells for antibiotic assay. App Microbiol. 16:1611-1612.

Bergsson G, Steingrimsson O, Thormar H. 2002. Bactericidal effects of fatty acids and monoglycerides on Helicobacter pylori. Int. J Antimicrob Agents 20:258-262.

Boney B, Hooper J, Parisot J. 2008. Principles of assessing bacterial susceptibility to antibiotics using the agar diffusion method. J Antimicrob Chemother. 61:1295-1301.

Friedman CR, Hoekstra RM, Samuel M, Marcus R, Bender J, Shiferaw B, Reddy S, Ahuja SD, Helfrick DL, Hardnett F, Carter M, Anderson B, Tauxe RV. 2004. Risk Factors for Sporadic Campylobacter Infection in the United States: A Case-Control Study in FoodNet Sites. Clin Infect Dis. 38(Suppl 3), S285-296.

Hermans D, Martel A, Van Deun K, Verlinden M, Van Immerseel Garmyn FA, Messens W, Heyndrickx M, Haesebrouck KF, Pasmans F. 2010. Intestinal mucus protects Campylobacter jejuni in the ceca of colonized broiler chickens against the bactericidal effects of medium-chain fatty acids. Poult Sci. 89:1144-1155.

Hinton A Jr. & Ingram KD. 2003. Bactericidal activity of tripotassium phosphate and potassium oleate on native flora of poultry skin. Food Microbiol. 20:405-410.

Hinton A Jr. & Ingram KD. 2005. Microbicidal activity of tripotassium phosphate and fatty acids towards spoilage and pathogenic bacteria associated with poultry. J Food Prot. 68:1462-1466.

Hinton A Jr., Cason JA, Buhr RJ, Liljebjelke K. 2009. Bacteria recovered from whole-carcass rinsates of broiler carcasses washed in a spray cabinet with lauric acid-potassium hydroxide. Int J Poult Sci. 8:1022-1027.

Kabara JJ, Vrable R, Lie Ken Jie MSF. 1977. Antimicrobial lipids: Natural and synthetic fatty acids and monoglycerides. Lipids 12:753-759.

Oyarzabal OA. 2005. Reduction of Campylobacter spp. by commercial antimicrobials applied during the processing of broiler chickens: A review from the Unites States Perspective. J Food Prot. 68:1752-1760.

Russell S & Keener K. 2007. Chlorine-misunderstood pathogen reduction tool. Poult Int. 46:24-30.

Solis de Los Santos F, Donoghue AM, Venkitanaryanan K, Reyes-Herrera I, Metcalf JH, Dirain ML, Aguiar RVF, Blore PJ, Donoghue DJ. 2008. Therapeutic supplementation of caprylic acid in feed reduces Campylobacter jejuni colonization in broiler chicks. Appl Environ Microbiol. 74:4564-4566.

Thorney MJ & Sleytr UB. 1974. Freeze-etching of the outer membranes of Pseudomonas and Acinetobacter. Arch. Microbiol. 100:409-417.