Comparing Organic Acids and Salt Derivatives as Antimicrobials Against Selected Poultry-Borne Listeria monocytogenes Strains In Vitro

Abstract

This article reports on the antilisterial properties of selected organic acids and salt derivatives in order to suggest possible alternatives in food preservation and pathogen control in the poultry meat processing industry. The susceptibility of two Listeria monocytogenes isolates was assessed against five organic acids (lactic, acetic, malic, citric, and propionic) and two acid-salt derivatives (sorbic acid [potassium salt] and benzoic acid [sodium salt]) across a series of pH environments. Minimum inhibitory concentrations (MICs) of the acids were tested against the two strains by means of an agar-dilution method. In general, strain CC60 was found to be more resistant than strain CC77 to both organic acids and salts. At pH values of 7 and above, high MIC levels (low susceptibility) were noted for potassium sorbate, sodium benzoate, and lactic acids, whereas susceptibility at lower pH increased reaching pH5 where the isolates were susceptible to all the organic acids tested. A small increase in pH notably reduced antimicrobial activity against the organisms. At pH 7, the isolates just about lost susceptibility to benzoic, lactic, malic, and sorbic acids. Although the activity of the majority of acids was linked to pH, some acids were not as closely related (e.g., potassium sorbate, sodium benzoate, and citric acid), and this suggests that the type of organic acids plays a role in inhibition. The relatively high MICs reported for compounds that are conventionally used as preservatives against Listeria spp. raise concern. The results furthermore suggest that the type of organic acid used to set pH, and not only pH alone, plays a role in determining inhibition. It was confirmed that a “one size fits all” approach to preservation is not always effective. Furthermore, the need for microbiological data to the subspecies level to inform the selection of preservatives was highlighted.

Introduction

F oodborne listeriosis is a growing concern amongst individuals with challenged immune systems such as pregnant women and people suffering from malnutrition and HIV/Aids (Farber and Peterkin, 1991; Nieman and Lorber, 1980; Ortel, 1989). Recently, media reports have drawn attention to outbreaks caused by L. monocytogenes in the United States, which left 16 people deceased and 72 infected across 18 states. This incident has again alerted the food safety industry to the ever-existing risks associated with foodstuffs such as poultry, cantaloupes, deli meats, fish, raw milk, and soft cheeses (Farber, 1993; Farber and Peterkin, 1991). Listeriosis is a prominent foodborne infection (Farber and Peterkin, 1991; Gellin et al., 1991; Kerr et al., 1993; Lennon et al., 1984; Riedo et al., 1990; Schlech et al., 1983) originating from unsatisfactory handling and processing, and has been demonstrated in several South African reports (Lues et al., 2007; Nel et al., 2004; Van Nierop et al., 2005). The ability of L. monocytogenes to grow at refrigeration temperatures emphasizes the importance of treatment with effective preservatives with bacteriostatic and bactericidal properties (Bremer and Osborne, 1995).

Buchanan et al. (1993) and Carrol et al. (2007) found L. monocytogenes to be sensitive to pH, and for this reason, organic acids have become popular as preservatives (Nakai and Siebert, 2003; Theron and Lues, 2007). Organic acids occur naturally in various foodstuffs and exhibit optimum antimicrobial activity at low pH levels that exclude most bacterial growth. When acting as food preservatives, organic acids exist in a pH-dependent equilibrium between the undissociated and dissociated states that is primarily responsible for the antimicrobial activity at a low pH (Brul and Coote, 1999; Nazer et al., 2005). The inhibitory action of an organic acid is further ascribed to its ability to freely cross the plasma membrane of bacterial or fungal cells (Brul and Coote, 1999). Once inside, the molecule dissociates and releases charged anions and protons that are toxic and inhibit essential microbial metabolic reactions (Eklund, 1985; Krebs et al., 1983; Russel, 1992). In this regard, Carroll et al. (2007) reported that acetic acid exhibits a greater antilisterial effect than lactic acid, since the latter cannot readily penetrate the cell membrane (Alvarado and McKee, 2007). Various researchers have found the antimicrobial properties of organic acids to be closely linked with pH, as well as differing in their mode of antimicrobial activity (Brul and Coote, 1999; Buchanan et al., 1993; Carroll et al., 2007; Piper et al., 2001; Stratford and Anslow, 1998).

Although preservatives such as sorbic acid have been used in the food industry as a standard microbial growth inhibitor, the development of resistance against organic acids and the resulting risk have been highlighted in recent times (Brul et al., 2002; Schnürer and Magnussen, 2005; Theron and Lues, 2007). In order to overcome the ability of organisms to adapt to the presence of weak monocarboxylic organic acids, sorbic and benzoic acids have been added in combination with other antimicrobials (Hazan et al., 2004; Marín et al., 2003). The development of resistance often arises from the exposure of L. monocytogenes to non-inhibitory concentrations of organic acids, which has been reported to induce an acid tolerance response and mutant strains of the organism that exhibit increased tolerance to low pH, and other stressors have been identified (Gahan et al., 1996; O'Driscoll et al., 1996).

The decreasing effectiveness of traditional preservatives and the global shift to organic products have necessitated investigations into alternative and novel compounds. The objective of the study reported in this article was to assess the antimicrobial profile of different organic acids and selected acid salt derivatives against two L. monocytogenes strains isolated from the poultry environment under different conditions. In addition, the suitability of the organic acids as food preservatives, as well as the extent to which antibacterial activity is influenced by pH was commented on. The article attempts to contribute to the body of knowledge regarding novel preservatives for the control of acid-tolerant and emerging foodborne pathogens.

Materials and Methods

Susceptibility testing

Isolates consisted of two Listeria monocytogenes strains that were collected, purified, and identified at a local poultry abattoir during various stages of processing. Partial 16S rRNA sequence data was compared to the National Center for Biotechnology Information database using BLAST algorithm, and the isolates (labeled CC60 and CC77) showed 100% and 99% similarity to Listeria monocytogenes strain American Type Culture Collection 19116 (JF967621.1), respectively. Organic acids commonly occurring in various foodstuffs (either naturally or as additives) were obtained from MP Biomedicals, Inc. (Solon, Ohio; sorbic acid [potassium salt], benzoic acid [sodium salt], lactic acid, acetic acid, malic acid, citric acid and propionic acid). The concentrations tested were based on current use as antimicrobials and their reported occurrence in a variety of foodstuffs.

Minimum inhibitory concentrations (MICs) of the organic acids were determined for the bacterial isolates via an agar-dilution method as described by the Clinical and Laboratory Standards Institute (CLSI, 2006a,b). Cell suspensions with a turbidity equivalent to a 0.5 McFarland standard (1.5×10⁸ CFU/mL) were prepared in sterile saline (0.85% NaCl) from overnight cultures incubated in Mueller-Hinton (MH) broth (Biolab Diagnostics Pvt. Ltd., Mumbai, India). The suspensions were re-diluted in sterile saline (1/10) and inoculated onto the agar surfaces using a multipoint inoculator (Mast Laboratories, Merseyside, UK) to deliver an inoculum of 3 μL (1×10⁵ CFU per spot). After 24-h incubation at 25°C, MICs were recorded as the lowest concentration of organic acid at which no growth was detected. Due to the lack of recorded susceptibility breakpoint concentrations for organic acids, the MICs were compared to reports from similar studies, as well as with commercial application guidelines.

Minimum inhibitory concentrations at different pH levels

The agar plates contained concentrations of organic acids in doubling increments (0.5, 1, 2, 4, 8, 14, 32, 64, 128 mM). The final pH concentrations ranged from pH 5 to pH 8 with 0.5 intervals (if required, minor adjustments to rounded pH values were done with 0.1 M HCl or 0.1 M NaOH). The control plates were adjusted with HCl or NaOH to the same pH values (5–8) but contained no organic acids. All results represent the means of at least triplicate analyses.

Results and Discussion

Susceptibilities of the L. monocytogenes strains (CC60 and CC77) are represented in Table 1, depicted as the MICs of all seven organic acids and salts for each strain at various pH levels. In the case of strain CC60, potassium sorbate and sodium benzoate showed high MICs, although lactic acid showed the highest total MIC value (512.5) across all pH environments. Lactic acid showed high MICs at lower pH values compared to the other compounds. The highest individual MICs (>128) were observed for potassium sorbate across predominantly higher pH levels, followed by sodium benzoate and lactic acid. Lactic acid demonstrated the lowest MIC (0.5) at pH 5. In the case of CC77, similar trends concomitant to change in pH were observed, although differences occurred amongst the various MICs.

Table 1.

Minimum Inhibitory Concentrations (MICs) of Listeria monocytogenes CC60 and CC77 at Various pH Levels

	MIC (mM) at various pH values
	Strain CC60
Organic acid or salt	pH 5.0	pH 5.5	pH 6.0	pH 6.5	pH 7.0	pH 7.5	pH 8.0
Potassium sorbate	1	8	16	64	128	>128	>128
Sodium benzoate	1	8	16	64	128	>128	>128
Lactic acid	0.5^a	32	32	64	128	128	128
Acetic acid	1	1	8	16	16	16	16
Citric acid	1	8	8	16	16	16	16
Malic acid	8	8	8	32	32	32	32
Propionic acid	2	8	16	16	16	32	32
	Strain CC77
Potassium sorbate	0.5	32	128	>128	>128	>128	>128
Sodium benzoate	0.5	8	32	128	>128	>128	>128
Lactic acid	0.5	32	64	64	64	128	128
Acetic acid	0.5	4	16	16	16	32	32
Citric acid	0.5	8	8	8	16	16	16
Malic acid	0.5	8	16	32	16	32	32
Propionic acid	0.5	8	8	16	16	32	32

No growth at the lowest concentration tested; this may imply that the MIC value could be lower than 0.5 mM.

In general, the lowest MICs across the pH range of 5–8 were observed for acetic and citric acids. This finding suggests that these acids were generally the most effective against the strains tested. Due to the insolubility of sorbic and benzoic acids, many applications use the salts (e.g., potassium sorbate and sodium benzoate) as preservatives (Padilla-Zakour, 1998). Sorbic acid is one of the more popular preserving acids, as it is not as dependent on pH and exhibits inhibitory activity at higher pH levels (pH 6.0–6.5) than would normally be expected from weak acids (Guynot et al., 2005; Stratford and Anslow, 1996). Sorbic acid also has less residual taste than other preservatives. However, Table 1 shows that, of all the compounds tested, one of the highest MIC values was recorded for sorbic acid salt.

In terms of pH, the most effective inhibitory activity against the isolates was demonstrated at pH 5. Strain CC77 did not grow at this pH in any of the organic acids tested. However, with a relatively small increase in pH, a notable decrease in antimicrobial activity was detected. At higher pH environments, the isolates exhibited a considerable degree of resistance to sodium benzoate, lactic acid, and potassium sorbate. The current study suggests that, not pH alone but also the type of acids and other ingredients in, for example, marinades, are similarly important in the successful control of Listeria. Combinations of preservatives and spices have been shown to exhibit antilisterial properties (Alvarado and McKee, 2007; Calicioglu et al., 2003; Carrol et al., 2007; Theron and Lues, 2007), by acting synergistically, even when present in lower singular concentrations.

The two strains of L. monocytogenes differed markedly in terms of their sensitivity to the various treatments, both in terms of pH (compared in terms of lactic acid at pH 5) and overall sensitivity. Across the board, CC60 was seen to be more resistant to organic acids and salt derivatives. Strain CC77 showed more resistance at low pH levels against lactic acid, whereas at pH levels of 6 and higher the MICs displayed by this organism rose rapidly towards surpassing (at about pH 5.5) that of CC60.

Conclusion

One should be prudent in predicting L. monocytogenes behavior in specific food products or environmental situations based on results obtained from in vitro laboratory simulations, since factors such as growth stage, pH, type of foodstuff, temperature, and prior exposure to non-inhibitory concentrations of preservatives should be taken into consideration. Nonetheless, if the MICs of strains that have not been exposed to food preservatives are as high as reported in this article, the actual situation with regard to MICs of Listeria spp. isolated from processed food raises concern. The findings emphasize the fact that a “one size fits all” approach when implementing preservation regimes is not a particularly effective approach to curb L. monocytogenes contamination in poultry plants. It may become imperative to establish standardized, user-friendly tests to monitor the susceptibility of L. monocytogenes strains against commonly used preservatives, in addition to elucidating the two-way role of pH and type of organic acid. It should further be determined whether L. monocytogenes lose susceptibility as a result of the influence of pH or becomes acid-tolerant as a result of ineffectiveness of organic acids as antimicrobial agents. The findings reported in this study indicated that the type of organic acid used to set pH, and not only pH alone, plays a role in determining the inhibition of Listeria spp. In order to shed light on the inhibition of re-emerging and resistant foodborne contaminants, further investigations into new and amended preservation regimes with novel antimicrobials should become a priority.

Footnotes

Acknowledgments

We wish to thank the National Research Foundation of South Africa for project funding.

Disclosure Statement

No competing financial interests exist.

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