Organic Acids: Chemistry. Antibacterial Activity and Practical Applications C. A . CHERRINGTON.'M . HINTON.'G . C. MEAD' and I . CHOPRA~ *Department of Veterinary Medicine. University of Bristol. Langford House. Langford. Avon BS18 7DU. U K . and bDepartment of Microbiology. University of Bristol. School of Medical Sciences. University Walk. Bristol. BS8 I TD. UK
I . Introduction
I1. Chemistry .
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. . A . Nomenclature B . Chemistryofthecarboxylgroup . . . . . 111. Antibacterial activity . . . . . . . . . A . Experimental conditions . . . . . . . B . Accumulationandmetabolism . . . . . . C . General observations . . . . . . . . D . The cell membrane . . . . . . . . E . Enzyme activity . . . . . . . . . F. Macromolecule synthesis . . . . . . . G . DNA . . . . . . . . . . . H . Recovery from and resistance to inhibition . . . IV . Practical applications . . . . . . . . . A . Animal-feedadditives . . . . . . . . B . Treatment of carcass meat and eggs . . . . . C. Other human foods, cosmetics and pharmaceuticals . V . Concludingremarks . . . . . . . . . References . . . . . . . . . . . .
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I Introduction
This review is concerned principally with the chemistry and antimicrobial activity of the saturated straight-chain monocarboxylic acids (Table 1). although reference is made. where appropriate. to derivatives of this group. e.g. unsaturated (cinnamic. sorbic). hydroxylic (citric. lactic). phenolic ADVANCES IN MICROBIAL PHYSIOLOGY. VOL . 32 ISBN (!-I24277324
Copyright0 1991. by AcademicPressLimited All rights ofreproductionin any form reserved
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TABLE 1. Nomenclature of organic acids (after Streitwieser and Heathcock 1981) Compound
Common name
Systematic name
short-chain fatty acids CI HCOOH Cz CH3COOH C3 CH1CHZCOOH (74 CH3(CHZ)ZCOOH Cs CHj(CHp)3COOH Ch CH3(CH2)4COOH
Formic Acetic Propionic Butyric Valeric Caproic
Methanoic Ethanoic Propanoic Butanoic Pentanoic Hexanoic
medium-chain fatty acids C7 CHdCHz)sCOOH Cn CH3(CH2)hCOOH Cg CH3(CHZ),COOH Cio CH~CHZ)&OOH
Enan thic Caprylic Pelargonic Capric
Heptanoic Octanoic Nonanoic Decanoic
(benzoic, cinnamic, salicylic) and multicarboxylic (azelaic, citric, succinic) acids (Table 2). For micro-organisms, organic acids can act either as a source of carbon and energy, or as inhibitory agents, depending on the concentration of the acid, its ability to enter the cell and the capacity of the organism to metabolize the acid. Organic acids and their salts have been employed for many years as preservative agents in food, drink and pharmaceutical products. Theories to explain their antimicrobial activity were reported as early as 1906 by Winslow and Lockeridge but, despite the increase in our understanding of microbial physiology and biochemistry, the mode of action of organic acids against micro-organisms still has not been satisfactorily explained. 11. Chemistry A . NOMENCLATURE
Organic acids are distinguished from other acids by the functional group C O O H to which an organic group or a hydrogen atom may be attached. Common names used to describe this group of organic compounds include fatty, volatile fatty, lipophilic, weak or carboxylic acids. The saturated
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ORGANIC ACIDS
TABLE 2. Formulae of common derivatives of the saturated straight-chain monocarboxylic acids Name
Formula
~~
Sorbic Citric Succinic Lactic Benzoic Cinnamic Azelaic Salicylic
CH,CH=CHCHCH=CHCOOH CH,COOHC( 0H)COOHCHzCOOH COOHCH2CHZCOOH CHRCH(0H)COOH C6HsCOOH C,HSCH=CHCOOH COOH(CH&COOH C,H,(OH)COOH
straight-chain organic acids listed in Table 1 may also be grouped arbitrarily according to their carbon-chain length, i.e. short-chain fatty acids (SCFA), medium-chain fatty acids (MCFA) and long-chain fatty acids (LCFA), which contain 1-6, 7-10 and 11 or more carbon atoms, respectively. The individual acids are named systematically from the normal alkane of the same number of carbon atoms, by dropping the final “e” and adding the suffix “oic” (Table 1). However, since some of the naturally occurring acids have been known for centuries, their common names (Table 1) are more familiar and these will be used in this review. B . CHEMISTRY OF THE CARBOXYL GROUP
In solid and liquid phases, organic acids exist predominantly in the dimeric form. Low-molecular-weight acids are liquid at room temperature, and the first four in the series, formic to butyric acids, are miscible with water. As chain length increases, water solubility decreases. However, MCFA readily form acid salts which are soluble in water. Organic acids are weakly acidic since they do not readily donate protons in aqueous solution. The relative strength of an acid is reflected in its dissociation constant K,, or pK, (-log Ka); the values for some acids are given in Table 3. The acid (HA) dissociates in water to the proton (H+) and anion (A-) such that at equilibrium [H+][A-]/[HA] = K,, the dissociation constant, which, because it is a ratio, is independent of acid concentration. Dissociation of weak acids is pH dependent and increases as pH values approach neutrality. The proportion of undissociated acid present at any pH value can be calculated from the formula [H+]/([H+] K,) (Lueck, 1980). Formic acid (pK, 3.7) is, therefore, a stronger acid than propionic acid (pK, 4.85), such that at most pH values it will have a smaller proportion of the acid in the undissociated form (Fig. 1). Salts of weak acids dissociate completely in aqueous solutions.
+
90 TABLE 3.
C. A. CHERRINGTON ET A L
pK, values of some organic acids (after Sheu eral., 1975, and Baird-Parker, 1980) Acid
PKa
Formic Acetic Propionic Butyric Pentanoic Hexanoic Octanoic Decanoic Palmitic Citric Lactic Sorbic Benzoic Cinnamic
3.7 4.8 4.9 4.9 4.9 4.9 4.9 4.9 5.1 3.1 3.1 4.8 4.2 4.4
PH
FIG. 1 . The relationship between pH value and dissociation of weak organic acids. Calculatedfrom Lueck (1980) (Section 1I.B). Key: 0, formic acid; 0,propionic acid.
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III. Antibacterial Activity A. EXPERIMENTAL CONDITIONS
There is no standard protocol for studying the antimicrobial activity of organic acids and many reports give only the pH value of the solution under test without indicating the concentration of the acid, thus making evaluation of the results difficult or impossible. In addition, it is difficult to compare the activity of different acids since it is influenced by the physical chemistry of each, the microbial species and the growth conditions, including the phase of growth. I . The Acid
Since, for any given acid, the proportion of undissociated acid molecules at any pH value is dependent on the pK, value, a direct comparison between acids is only possible if their pK, values are the same. Otherwise, to obtain the same number of undissociated acid molecules, the pH values of equimolar solutions would have to be varied as appropriate. Differences in acid activity will also depend on the buffering capacity of the medium, the presence of organic compounds, e.g. casein in acid milk products (Rubin, 1985), the acid concentration (Cowles, 1941; Barker, 1964; Baskett and Hentges, 1973; Freese et al., 1973; Przybylski and Witter, 1979; Eklund, 1980), the structure of the acid, e.g. chain length and saturation (Reid, 1932; Bergeim, 1940; Kabara et al., 1972), and whether acid salts or mixtures of acids are used (Minor and Marth, 1972; Adams and Hall, 1988). 2. The Micro-organism
Gram-negative bacteria are relatively resistant to MCFA and LCFA (see Section 1II.C) while the ability of the organism to metabolize SCFA may affect the antibacterial activity of the acid. Another consideration is the form in which the cells are tested, i.e. as whole cells, protoplasts or membrane vesicles. This is particularly relevant when LCFA are being studied, since they are lipophilic molecules and their antimicrobial activity is probably related to their ability to become integrated with the cell membrane (Greenway and Dyke, 1979). There is evidence that exposure of bacteria, e.g. Escherichia coli and salmonellas, to sublethal acidic conditions increases their acid tolerance (Huhtanen, 1975; Goodson and Rowbury 1989a,b) and this favours their subsequent survival in environments with lethal pH values. This resistance is of potential importance since it may allow harmful or unwanted
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acid-habituated organisms to survive in acid foods or at body sitessuch as the urinary tract and vagina. It is lost quickly after transfer to neutral media (Huhtanen, 1975), although there may be an increased lag time before growth recommences (Minor and Marth, 1972). The mechanism of this resistance is not known but it is not due to sensitization to hydrogen peroxide (Goodson and Rowbury, 1989a).
3. The Culture Conditions The culture conditions, e.g. pH value, temperature, water activity and aeration, affect the growth rate of the organism and these, in turn, influence the response to the acid (Fay and Farias, 1975,1976; Meyer etal., 1981). In general, any factor which reduces growth rate increases the sensitivity of the cell to acids while bacteria in the stationary phase are more sensitive to the presence of organic acids than those in the log phase of growth (Fay and Farias, 1975, 1976; Chemngton et d.,1991a), although the basis for this increased sensitivity has not been established. In addition, the dissociation of the acid is affected by both pH value and temperature, thereby influencing the concentration of undissociated acid in the medium. 4. The Composition of the Medium
Nutrient broth protects E. coli against the activity of organic acids while inhibition may be less when the bacterial cells, e.g. Bacillus subtilis, are grown on a minimal medium because they are less sensitive to the acids when growing slowly (Sheu and Freese, 1972). On the other hand, incubation in buffer enhances acid activity (Tsuchido et al., 1985; C. A. Cherrington, unpublished observations). The choice of diluent used when determining the number of viable cells will also affect the final result, since buffers at neutral pH result in higher counts than buffers at acid pH values (Sinha 1986), presumably because the cells can more readily repair acid-induced injuries at “benign” pH values. Finally, the composition of the medium used to culture acid-treated cells is also important since fewer cells are recovered on selective than on unselective media, e.g. violet-red bile agar for E. coli (Roth and Keenan, 1971; Przybylski and Witter, 1979) and trypticase soy agar with 7% NaCl for Staphylococcus aureus (Zayaitz and Ledford, 1985). A practical consequence of these observations is that selective media can be used to estimate the proportion of injured cells in a population (Pryzbyslki and Witter, 1979; Blankenship, 198l), but they should not be used for enumerating bacteria present in acid foods.
ORGANIC ACIDS
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B. ACCUMULATION AND METABOLISM
1. Long- and Medium-Chain Organic Acids Gram-negative bacteria are capable of metabolizing LCFA and MCFA. The uptake and metabolism of these acids in E. coli has been reviewed by Nunn (1987). LCFA and MCFA traverse the membrane via porins, bind to the fudL gene product to cross the inner membrane and are concurrently activated by the fadD gene product, acyl-CoA synthetase. Inside the cell, the activated fatty acid is degraded via the (3-oxidationcycle, whose enzymes are induced by the activated acid. A specific uptake system for organic acids has not been reported for Gram-positive bacteria and they are thought instead to become integrated with the lipid bilayer of the cell membrane (Greenway and Dyke, 1979). 2. Short-Chain Organic Acids
The mode of entry of SCFA into the microbial cell has not been established, although it is assumed that only the neutral (undissociated) acid molecule enters the cell (Baskett and Hentges, 1973; Cramer and Prestgard, 1977; Chu et al., 1987). Since the acid molecules are lipid soluble, it has been presumed that they diffuse freely across the cell membrane (Salmond et al. , 1984; Nunn, 1987). However, a number of reports suggest that energylinked carriers and the membrane potential (described in Section III.D.2) may be involved in their uptake, e.g. acetoacetyl-CoA transferase and ATP are required for E. coli to grow on butyrate (Vanderwinkel et al., 1968) and for butyrate and acetate uptake in Clostridium acetobutylicum (Wiesenborn et al., 1989). Salmonella spp. transport citrate via a specific permease (Sommers et al., 1981) while, in methylotrophic bacteria, a pH difference (ApH) across the cell membrane is required to accumulate formic acid (Chu et al., 1987). Bacteria can utilize acetic acid as a carbon and energy source by inducing enzymes of the glyoxylate pathway, isocitrate lyase and malate synthase, which allows net assimilation of carbon (Nunn, 1987). Escherichia coli is also able to utilize propionic acid as a sole carbon and energy source (Wegener et al., 1968) and, although Kay (1972) identified two routes for propionate metabolism, a-oxidation to pyruvate and metabolism via hydroxyglutarate, the precise pathway is not known (Nunn, 1987). Growth in the presence of calcium propionate results in synthesis of fatty acids of unusual composition, namely saturated CI5and CI7and unsaturated CI7fatty acids, probably as a result of using propionyl-ACP as a primer for fatty acid synthesis instead of acetyl-ACP (Ingram, 1977).
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C. A. CHERRINGTON ET Al..
C. GENERAL OBSERVATIONS
The following section considers published reports which deal with the effects of organic acids on microbial growth. For reviews of specific acids see Bosund (1962; benzoic, salicylic), Sofos and Busta (1981; sorbic) and Sofos et al. (1986; sorbic), while for a general review of the mode of action of various compounds used as preservatives and antiseptics see Freese and Levine (1978). Organic acids are more effective than mineral acids as antimicrobial agents (e.g. Nunheimer and Fabian, 1940; Reynolds, 1975) and, although they exhibit broad-spectrum antimicrobial activity, the antibacterial efficiency of individual acids varies (Nunheimer and Fabian, 1940; Cowles, 1941; Barker, 1964; Goepfert and Hicks, 1969; Minor and Marth, 1972; Sheu et al., 1975). For instance, in a study involving Staphylococcus aureus, Minor and Marth (1972) found that acetic and lactic acids were more active than hydrochloric acid and, while mixtures of hydrochloric and lactic acids were superior to either alone, there was no additive effect when acetic and hydrochloric acids were mixed together. Vegetative cells are more sensitive to organic acids than the corresponding spore forms (Wong and Chen, 1988) with bacteria showing increasing sensitivity to acids as concentration and (with the exception of Gramnegative species) chain length increase (Reid, 1932; Cowles, 1941; Barker, 1964; Freese et al., 1973; Sheu et al., 1975; Eklund, 1980; Brackett, 1987). Although Gram-negative bacteria can actively transport these acids into the cell and metabolize them via the P-oxidation cycle, Sheu and Freese (1973) showed that resistance was caused by the lipopolysaccharide layer in the cell wall, which prevents the acids from entering the cell. The antimicrobial activity of organic acids increases with decreasing pH value (Cohen and Clarke, 1919; Bosund, 1962; Hentges, 1967; Freese etal., 1973) and, since a greater proportion of undissociated molecules exist as the pH value decreases (see Section II.B), it has been assumed that it is the undissociated molecule that is the antimicrobial agent (e.g. Reid, 1932; Bergeim, 1940; Cowles, 1941; Barker, 1964). However, this takes no account of the behaviour of the acid inside the cell. The pH value of the cytoplasm of bacteria is thought to be regulated strictly, with estimates ranging from 7.4 to 7.6 (Padan et al., 1981; Slonczewski etal., 1981) and 8.2 to 8.7 (Lagarde, 1977; Booth etal., 1979) over an external pH range of 5.59. This means that once the acid molecules have entered the cell (Freese et a l . , 1973; Salmorid et al., 1984) they will dissociate almost totally, assuming that dissociation of the acid in cytoplasm is the same as in aqueous solutions, and it is probable that the proton and anion both contribute to inhibition of growth of the bacteria (Rubin et al., 1982; Eklund, 1983,1985; Salmond ec al., 1984; Cherrington, et al., 1990a). Indeed, Eklund (l983,1985),’using a
ORGANIC ACIDS
95
mathematical model, calculated that the anions of benzoic, propionic and sorbic acids contributed to more than 50% of the growth inhibition of E. cofi in a medium of pH 6 or more. D.
THE CELL MEMBRANE
The cell membrane has been identified as one of the potential sites of action of organic acids. Other “targets” include enzymes (Section III.E), macromolecule synthesis (Section 1II.F) and DNA (Section 1II.G). I . Cell-Membrane Integrity
Organic acids have been regarded as anionic surfactants acting as membrane-disrupting disinfectants (Sheu and Freese, 1972; Kondo and Kanai, 1976; Greenway and Dyke, 1979). However, cell lysis has only been reported with LCFA where the protective cell wall has been removed, e.g. protoplasts of Bacillus megaterium and Micrococcus lysodeikticus (Galbraith and Miller, 1973) and membrane vesicles of Bacillus subtilis (Freese et al., 1973). In contrast, LCFA did not induce lysis in the cell-walllacking mycobacteria (Kondo and Kanai, 1976) and 0.5 M formic or propionic acids, buffered to pH 5, did not disrupt E. coli cell membranes (Cherrington et al., 1991a). Statham and McMeekin (1988) showed that the addition of lyzozyme was required to produce lysis in Alteromonas putrefaciens treated with sorbate while Tsuchido et al. (1985) proposed that dodecanoic acid induced autolytic enzymes in B. subtilis. Interference with membrane permeability, characterized by leakage of cellular proteins or ions (e.g. K’, Na+) has been reported for Gram-positive bacteria and mycobacteria incubated with LCFA (Galbraith and Miller, 1973: Kondo and Kanai, 1976; Greenway and Dyke, 1979) while Corlett and Brown (1980) suggested that weak lipophilic acids cause leakage of protons across the cell membrane, which then acidify the cytoplasm and inhibit nutrient transport. On the other hand, acetate buffer at pH 4.2 did not cause leakage of protein in E. colicultures (Przybylski and Witter, 1979) and at pH 3.2-3.5 produced only slight leakage in Salmonella bareilly cultures (Blankenship 1981). 2. Cell Membrane Functions
Minor disturbances of the cytoplasmic membrane which inactivate energylinked reactions can prevent growth. The selective permeability of the cytoplasmic membrane produces an ionic gradient (which drives the transport of certain substrates) and is involved in energy generation and
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regulation of the internal pH value (for a review, see Booth, 1985). This electrochemical proton gradient ApH+ (also known as the proton-motive I a proton gradient ApH force, or Ap) comprises an electrical gradient A ~ and as follows:
A p = A w - ZApH where Z = 2.3(RT/F)with R the gas constant in J K-’, Tthe thermodynamic temperature and F the Faraday constant In general, as external pH value decreases, Ap is maintained by increasing A ~toI compensate for the increase in ApH (Ramos and Kaback, 1977; Padan et al., 1981). The general mechanism of microbial growth inhibition by organic acids was considered to be the acidification of the cell cytoplasm as a result of the release of excess protons following dissociation of the acid (Baird-Parker, 1980). Salmond et al. (1984) compared the effect of acid concentration on cytoplasmic pH (internal pH or pHi) and demonstrated a lowering of the pHi and a reduced growth rate as acid concentration increased. However, it was concluded that the drop in pHi alone was unlikely to be the primary cause of growth inhibition, and a synergistic effect of H+ and accumulated undissociated acid was suggested. By definition, acidification of the cytoplasm would reduce the ApH and, consequently, organic acids have been termed “uncoupling agents” because they rapidly shuttle protons across the membrane, thereby dissipating the proton-motive force (Finkelstein, 1970; Levin and Freese, 1977; Freese and Levin, 1978; Baronofsky et al., 1984; Herrero et al., 1985). This claim is supported by reports that Ap-driven substrate-transport systems are inhibited in the presence of organic acids (Freese et al., 1973; Sheu et al., 1975; Eklund, 1980). However, Eklund (1985) showed that sorbic acid abolished ApH, but not Ay, and since A ~ provides I a sufficient energy gradient on its own to “drive” the uptake of several substances in E. coli (e.g. cysteine, leucine, proline and succinate; Ramos and Kaback, 1977) concluded that inhibition of substrate transport was not the primary target of organic acids. ATP levels in B. subtilis and E. coli are decreased following incubation with organic acids, although this is not a primary cause of growth inhibition (Sheu and Freese, 1972; Freese et al., 1973; Sheu et al., 1975; Cherrington, 1990). In addition, although organic acids inhibit oxidative metabolism in E. coli and B. subtilis, the concentration of acid needed to inhibit the process by 50% was higher than that required to inhibit bacterial growth (Weiner and Draskoczy , 1961).
ORGANIC ACIDS
97
E. ENZYME ACTIVITY
In general, the activity of enzymes is lowered by acid pH values, and this may be a secondary effect of the acidification of the cytoplasm. Octanoic acid selectively inhibits glucose-phosphate dehydrogenase, pyruvate kinase, fumarase and lactate dehydrogenase of Arthrobacter crystallopoietes (Ferdinandus and Clarke, 1969), although this may reflect the specific sensitivity of these enzymes to p H value. However, York and Vaughn (1964) reported that inhibition of the sulphydryl enzymes fumarase, aspartase and succinate dehydrogenase by sorbic acid was the result of the acid reacting with the thiol group of cysteine. Studies involving the yeast Cundida utilis showed that the respiratory rate and extracellular concentration of unused acetate were influenced by the pH value of the culture medium, with the toxicity of the acetate being due to the uncoupling of oxidative phosphorylation (Hueting and Tempest, 1977) while the inhibitory activity of organic acids for Thiobacillus ferrooxidans was due to the direct inhibition of the iron-oxidase system (Tuttle and Dugan, 1976). F. MACROMOLECULE SYNTHESIS
The sensitivity of individual biosynthetic functions to acids appears to vary depending on the bacterium and the acid. For example, potassium sorbate inhibits protein, RNA and DNA syntheses equally in Pseudomonas fruorescens (Nose, 1982), while protein synthesis is primarily inhibited by azelaic acid in Staphylococcus epidermidis (Bojar et al., 1988). Both formic and propionic acids were most active in vitro against DNA synthesis in E. coli, although lipid, peptidoglycan, protein and RNA syntheses were also inhibited (Cherrington et al., 1990a). Inhibition of macromolecule synthesis may reflect the sensitivity of the biosynthetic enzymes to acidification of the cytoplasm. However, Cherrington (1991a,b) suggested that the cytoplasmic pH value recovered in the presence of sublethal concentrations of formic acid in the absence of a corresponding recovery in the rate of DNA synthesis, although rates of lipid, peptidoglycan, protein and RNA syntheses showed some recovery. G . DNA
Sinha (1986) incubated stationary-phase DNA-repair-deficient mutants of E. coli with acetic and lactic acids (pH 3.5) and reported an increased sensitivity of the polA2 mutant (which lacked DNA polymerase activity) compared to its isogenic parent, implying that the acids caused physical damage to the DNA molecule. Stephens and Dalton (1987) also found that
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acids can damage DNA because benzoate, and to a lesser extent acetate and butyrate, induced both base deletions in To1 plasmids and plasmid curing in Pseudomonas putida, possibly by excision of a transposon-like region. In contrast, Cherrington et al. (1991b) were unable to detect an increased sensitivity of log-phase cultures of polAZ, recA56 or uvrA6 mutants to formic and propionic acids (pH 5) compared to their isogenic parent strain. Similarly, there was no evidence of an SOS response in E. coli, in which damage to the DNA molecule is manifested by the cell forming filaments (Little and Mount, 1982) after exposure to sublethal concentrations of the same acids. Cherrington (1990) suggested that the acid anion may interfere with the conformation of the DNA molecule by interacting with ion charges around it. The differences in anion structure could then explain the differences in activity of organic acids with the same, or similar, pKa values, e.g. lactic acid (pK, 3.1) is more active than citric acid (pK, 3.1), sorbic acid (pK, 4.8) is more active than propionic acid (pKa 4.9), and cinnamic acid (pKa 4.4) is more active than benzoic acid (pK, 4.2) (Sheu et al., 1975; Salmond et al., 1984; M. Hinton, unpublished observations). H. RECOVERY FROM AND RESISTANCE TO INHIBITION
Inhibition of organic acids appears reversible, since acid-treated cells will grow when placed in an acid-free medium (Przybylski and Witter, 1979; Blankenship, 1981), with recovery of Salmonella bareilly (Blankenship, 1981) and Sfaphy1ococcu.saureu.s (Zayaitz and Ledford, 1985) from acetic acid injury requiring both protein and RNA synthesis and an active respiratory (electron-transport) chain. Resistance of bacteria to organic acids has not been reported, although E. coli can adapt to utilize propionic acid (Wegener et al., 1968), acetic acid and LCFA (reviewed by Nunn, 1987) as sole carbon and energy sources. IV. Practical Applications
This section of the review will concentrate principally on two aspects of the use of organic acids: firstly, in animal husbandry as animal feed additives and, secondly, in abattoirs and food-processing plants where they may be used in controlling microbial contamination of carcass meat. The section will conclude with brief reference to their use in other human foods and in other products destined for human use.
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A. ANIMAL-FEED ADDITIVES
Silage is a feed material produced by the fermentation of high-moisture crops such as grass, legumes and maize. In order to produce a high-quality product it is essential that pH values of these crops are lowered as rapidly as possible after harvesting, and this process can be hastened by the addition of both inorganic and organic acids (Woolford, 1984) with formic acid being commonly used as either the sole ingredient or in combination with other chemicals. Woolford (1975) evaluated the straight-chain acids up to CI2and concluded that all had potential for use as silage additives. Formic and propionic acids were useful in aiding the production of conventional fermented silages; the higher acids, which are likely to be too expensive for commercial use, produced a silage which was not fermented, while some acids, e.g. butyric, valeric and caproic acids, would prove unacceptable because of their unpleasant smell. Organic acids have been added to animal feed as an alternative to heat treatment to control microbial contaminants (Francis and Turnbull, 1979), although they will not necessarily eliminate pathogens such as the salmonellas (Kahn and Katamay, 1969; Duncan and Adams, 1972;Vanderwal, 1979; Banton et al., 1984; Humphrey and Lanning, 1988). The efficiency of these agents as disinfectants can be increased by increasing their concentration (Vanderwal, 1979; van Staden et al., 1980; Rejholec, 1981). This approach may be justified when heavily contaminated ingredients are being used, although this may reduce palatability (Rys and Koreleski, 1974;Cave, 1984) unless the treated ingredient is diluted by subsequent mixing with other matrials. The addition of formic acid to the feed of laying hens lowered the incidence of salmonella infections in their newly hatched progeny, although this benefit did not carry through to slaughter because the chicks were given untreated feed (Humphrey and Lanning, 1988). Similarly, Hinton and Linton (1988) reported that a mixture of formic and propionic acids, which can be expected to have no ultimate effect on the organoleptic quality of the carcass meat (Basker and Klinger, 1979), effectively reduced salmonella colonization in chicks given contaminated acid-treated feed. However, it was still possible to isolate salmonellas from the acid-treated feed itself (Hinton and Linton, 1988), which suggests that the acids may exert their effect in the birds’ crops after the feed has been moistened following consumption. It is also necessary to give acid-treated feed throughout the rearing period, since the acids have no beneficial effect once the birds have become infected (Hinton and Linton, 1988). The acidification of water has also been proposed as a way of controlling salmonella infections. This procedure could reduce water consumption by young chicks since they appear less tolerant of water with acid pH values
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than are older birds (Fuerst and Kare, 1962), but may prove effective in reducing cross-infection via contaminated water. However, it was of no value in controlling feed-borne infections (Al-Chalaby et al., 1985). Acidified milk replacers are used to feed unweaned calves, and their use has been associated with a reduction in coliform numbers in milk prepared on the farm (Morgan-Jones and Hinks, 1980) and in the faeces of young calves fed these products (Simm et al., 1980; Humphrey er al., 1982). However, there is little evidence to suggest that they have any beneficial effect either nutritionally or for the control of enteric disease caused by E. cofi (Stobo, 1983). The acidification of the diets of young pigs has been reviewed by Easter (1988). The principal reason for incorporating acids (e.g. citric or lactic acid) in their feed was to minimize the check in growth rate which invariably occurs after weaning and is frequently associated with the development of diarrhoea. There is a substantial literature which indicates that pigs respond well to an acidified diet during the weeks following weaning, and Easter (1988) concluded that the greatest benefit occurred when the diets comprised cereal grains and plant proteins and contained no lactose, although Burnell et a f . (1988) have recorded improved performance with diets containing whey powder. B. TREATMENT OF CARCASS MEAT AND EGGS
Organic acids, especially acetic and lactic acids, have been widely studied as potential decontaminating agents for red meat, principally from cattle and sheep, and poultry carcasses. Most studies concerning red meat have involved either dipping the meat in a treatment solution, which requires frequent renewal to maintain activity (Eustace, 1984), or the use of spray systems that deliver a single or repeated application. In a study of lamb carcasses that involved spraying or dipping (Anderson et al., 1988) the meat was treated with 1.5 or 3.0% acetic acid at 25 or 55°C. Each treatment reduced counts significantly in comparison with untreated controls, with dipping in 3% acid at 55°C being the most effective. Hamby et al. (1987) treated beef carcasses by intermittent spray chilling using water, 1%acetic or lactic acid, or a single spray treatment with either acid. Intermittent spraying gave the best results, lowering total viable counts from portions of meat by 100,000-fold following storage of the meat in vacuum packs at 2°C for 28 days. Reductions observed 48 hours after treatment are shown in Table 4. For beef artificially contaminated with faecal bacteria and then dipped for 15 seconds in acetic acid at concentrations ranging from 1 to 3% and temperatures varying from 25 to 70°C, treatment with 3% acid at 70°C was
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TABLE 4. Mean total viable counts (log,&m2) from five sampling sites on beef sides receiving intermittent spraying with water or organic acid Sampling site
Water
Acetic acid
Lactic acid
Inside round
Control Treated Difference
2.9" 2.5" 0.4
2.3" 1 .5" 0.8
2.6" 1 .Sb 0.8
Strip loin
Control Treated Difference
2.2" 2.4" -0.2
3.4"
1.4"
2.0
2.6" 1 .s6 1.1
Boneless rib
Control Treated Diffefence
2.6" 3.8' -1.2
4.5" 2.16 2.4
3.4" 1.36 2.1
Clod
Control Treated Difference
3.0" 3.2" -0.2
2.8' 1.16 1.7
3.2" 1.26 2.0
Inside neck
Control Treated Difference
2.5" 2.1"
2.6" 1.8" 0.8
3.Y
0.4
1.56
2.4
Means represent data from four carcasses. Means within same sampling site and treatment with different superscripts differ significantly (P
I . Introduction
I1. Chemistry .
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. . A . Nomenclature B . Chemistryofthecarboxylgroup . . . . . 111. Antibacterial activity . . . . . . . . . A . Experimental conditions . . . . . . . B . Accumulationandmetabolism . . . . . . C . General observations . . . . . . . . D . The cell membrane . . . . . . . . E . Enzyme activity . . . . . . . . . F. Macromolecule synthesis . . . . . . . G . DNA . . . . . . . . . . . H . Recovery from and resistance to inhibition . . . IV . Practical applications . . . . . . . . . A . Animal-feedadditives . . . . . . . . B . Treatment of carcass meat and eggs . . . . . C. Other human foods, cosmetics and pharmaceuticals . V . Concludingremarks . . . . . . . . . References . . . . . . . . . . . .
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I Introduction
This review is concerned principally with the chemistry and antimicrobial activity of the saturated straight-chain monocarboxylic acids (Table 1). although reference is made. where appropriate. to derivatives of this group. e.g. unsaturated (cinnamic. sorbic). hydroxylic (citric. lactic). phenolic ADVANCES IN MICROBIAL PHYSIOLOGY. VOL . 32 ISBN (!-I24277324
Copyright0 1991. by AcademicPressLimited All rights ofreproductionin any form reserved
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C. A. CHERRINGTON EJ A L
TABLE 1. Nomenclature of organic acids (after Streitwieser and Heathcock 1981) Compound
Common name
Systematic name
short-chain fatty acids CI HCOOH Cz CH3COOH C3 CH1CHZCOOH (74 CH3(CHZ)ZCOOH Cs CHj(CHp)3COOH Ch CH3(CH2)4COOH
Formic Acetic Propionic Butyric Valeric Caproic
Methanoic Ethanoic Propanoic Butanoic Pentanoic Hexanoic
medium-chain fatty acids C7 CHdCHz)sCOOH Cn CH3(CH2)hCOOH Cg CH3(CHZ),COOH Cio CH~CHZ)&OOH
Enan thic Caprylic Pelargonic Capric
Heptanoic Octanoic Nonanoic Decanoic
(benzoic, cinnamic, salicylic) and multicarboxylic (azelaic, citric, succinic) acids (Table 2). For micro-organisms, organic acids can act either as a source of carbon and energy, or as inhibitory agents, depending on the concentration of the acid, its ability to enter the cell and the capacity of the organism to metabolize the acid. Organic acids and their salts have been employed for many years as preservative agents in food, drink and pharmaceutical products. Theories to explain their antimicrobial activity were reported as early as 1906 by Winslow and Lockeridge but, despite the increase in our understanding of microbial physiology and biochemistry, the mode of action of organic acids against micro-organisms still has not been satisfactorily explained. 11. Chemistry A . NOMENCLATURE
Organic acids are distinguished from other acids by the functional group C O O H to which an organic group or a hydrogen atom may be attached. Common names used to describe this group of organic compounds include fatty, volatile fatty, lipophilic, weak or carboxylic acids. The saturated
89
ORGANIC ACIDS
TABLE 2. Formulae of common derivatives of the saturated straight-chain monocarboxylic acids Name
Formula
~~
Sorbic Citric Succinic Lactic Benzoic Cinnamic Azelaic Salicylic
CH,CH=CHCHCH=CHCOOH CH,COOHC( 0H)COOHCHzCOOH COOHCH2CHZCOOH CHRCH(0H)COOH C6HsCOOH C,HSCH=CHCOOH COOH(CH&COOH C,H,(OH)COOH
straight-chain organic acids listed in Table 1 may also be grouped arbitrarily according to their carbon-chain length, i.e. short-chain fatty acids (SCFA), medium-chain fatty acids (MCFA) and long-chain fatty acids (LCFA), which contain 1-6, 7-10 and 11 or more carbon atoms, respectively. The individual acids are named systematically from the normal alkane of the same number of carbon atoms, by dropping the final “e” and adding the suffix “oic” (Table 1). However, since some of the naturally occurring acids have been known for centuries, their common names (Table 1) are more familiar and these will be used in this review. B . CHEMISTRY OF THE CARBOXYL GROUP
In solid and liquid phases, organic acids exist predominantly in the dimeric form. Low-molecular-weight acids are liquid at room temperature, and the first four in the series, formic to butyric acids, are miscible with water. As chain length increases, water solubility decreases. However, MCFA readily form acid salts which are soluble in water. Organic acids are weakly acidic since they do not readily donate protons in aqueous solution. The relative strength of an acid is reflected in its dissociation constant K,, or pK, (-log Ka); the values for some acids are given in Table 3. The acid (HA) dissociates in water to the proton (H+) and anion (A-) such that at equilibrium [H+][A-]/[HA] = K,, the dissociation constant, which, because it is a ratio, is independent of acid concentration. Dissociation of weak acids is pH dependent and increases as pH values approach neutrality. The proportion of undissociated acid present at any pH value can be calculated from the formula [H+]/([H+] K,) (Lueck, 1980). Formic acid (pK, 3.7) is, therefore, a stronger acid than propionic acid (pK, 4.85), such that at most pH values it will have a smaller proportion of the acid in the undissociated form (Fig. 1). Salts of weak acids dissociate completely in aqueous solutions.
+
90 TABLE 3.
C. A. CHERRINGTON ET A L
pK, values of some organic acids (after Sheu eral., 1975, and Baird-Parker, 1980) Acid
PKa
Formic Acetic Propionic Butyric Pentanoic Hexanoic Octanoic Decanoic Palmitic Citric Lactic Sorbic Benzoic Cinnamic
3.7 4.8 4.9 4.9 4.9 4.9 4.9 4.9 5.1 3.1 3.1 4.8 4.2 4.4
PH
FIG. 1 . The relationship between pH value and dissociation of weak organic acids. Calculatedfrom Lueck (1980) (Section 1I.B). Key: 0, formic acid; 0,propionic acid.
ORGANIC ACIDS
91
III. Antibacterial Activity A. EXPERIMENTAL CONDITIONS
There is no standard protocol for studying the antimicrobial activity of organic acids and many reports give only the pH value of the solution under test without indicating the concentration of the acid, thus making evaluation of the results difficult or impossible. In addition, it is difficult to compare the activity of different acids since it is influenced by the physical chemistry of each, the microbial species and the growth conditions, including the phase of growth. I . The Acid
Since, for any given acid, the proportion of undissociated acid molecules at any pH value is dependent on the pK, value, a direct comparison between acids is only possible if their pK, values are the same. Otherwise, to obtain the same number of undissociated acid molecules, the pH values of equimolar solutions would have to be varied as appropriate. Differences in acid activity will also depend on the buffering capacity of the medium, the presence of organic compounds, e.g. casein in acid milk products (Rubin, 1985), the acid concentration (Cowles, 1941; Barker, 1964; Baskett and Hentges, 1973; Freese et al., 1973; Przybylski and Witter, 1979; Eklund, 1980), the structure of the acid, e.g. chain length and saturation (Reid, 1932; Bergeim, 1940; Kabara et al., 1972), and whether acid salts or mixtures of acids are used (Minor and Marth, 1972; Adams and Hall, 1988). 2. The Micro-organism
Gram-negative bacteria are relatively resistant to MCFA and LCFA (see Section 1II.C) while the ability of the organism to metabolize SCFA may affect the antibacterial activity of the acid. Another consideration is the form in which the cells are tested, i.e. as whole cells, protoplasts or membrane vesicles. This is particularly relevant when LCFA are being studied, since they are lipophilic molecules and their antimicrobial activity is probably related to their ability to become integrated with the cell membrane (Greenway and Dyke, 1979). There is evidence that exposure of bacteria, e.g. Escherichia coli and salmonellas, to sublethal acidic conditions increases their acid tolerance (Huhtanen, 1975; Goodson and Rowbury 1989a,b) and this favours their subsequent survival in environments with lethal pH values. This resistance is of potential importance since it may allow harmful or unwanted
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C. A. CHERRINGTON ET A L
acid-habituated organisms to survive in acid foods or at body sitessuch as the urinary tract and vagina. It is lost quickly after transfer to neutral media (Huhtanen, 1975), although there may be an increased lag time before growth recommences (Minor and Marth, 1972). The mechanism of this resistance is not known but it is not due to sensitization to hydrogen peroxide (Goodson and Rowbury, 1989a).
3. The Culture Conditions The culture conditions, e.g. pH value, temperature, water activity and aeration, affect the growth rate of the organism and these, in turn, influence the response to the acid (Fay and Farias, 1975,1976; Meyer etal., 1981). In general, any factor which reduces growth rate increases the sensitivity of the cell to acids while bacteria in the stationary phase are more sensitive to the presence of organic acids than those in the log phase of growth (Fay and Farias, 1975, 1976; Chemngton et d.,1991a), although the basis for this increased sensitivity has not been established. In addition, the dissociation of the acid is affected by both pH value and temperature, thereby influencing the concentration of undissociated acid in the medium. 4. The Composition of the Medium
Nutrient broth protects E. coli against the activity of organic acids while inhibition may be less when the bacterial cells, e.g. Bacillus subtilis, are grown on a minimal medium because they are less sensitive to the acids when growing slowly (Sheu and Freese, 1972). On the other hand, incubation in buffer enhances acid activity (Tsuchido et al., 1985; C. A. Cherrington, unpublished observations). The choice of diluent used when determining the number of viable cells will also affect the final result, since buffers at neutral pH result in higher counts than buffers at acid pH values (Sinha 1986), presumably because the cells can more readily repair acid-induced injuries at “benign” pH values. Finally, the composition of the medium used to culture acid-treated cells is also important since fewer cells are recovered on selective than on unselective media, e.g. violet-red bile agar for E. coli (Roth and Keenan, 1971; Przybylski and Witter, 1979) and trypticase soy agar with 7% NaCl for Staphylococcus aureus (Zayaitz and Ledford, 1985). A practical consequence of these observations is that selective media can be used to estimate the proportion of injured cells in a population (Pryzbyslki and Witter, 1979; Blankenship, 198l), but they should not be used for enumerating bacteria present in acid foods.
ORGANIC ACIDS
93
B. ACCUMULATION AND METABOLISM
1. Long- and Medium-Chain Organic Acids Gram-negative bacteria are capable of metabolizing LCFA and MCFA. The uptake and metabolism of these acids in E. coli has been reviewed by Nunn (1987). LCFA and MCFA traverse the membrane via porins, bind to the fudL gene product to cross the inner membrane and are concurrently activated by the fadD gene product, acyl-CoA synthetase. Inside the cell, the activated fatty acid is degraded via the (3-oxidationcycle, whose enzymes are induced by the activated acid. A specific uptake system for organic acids has not been reported for Gram-positive bacteria and they are thought instead to become integrated with the lipid bilayer of the cell membrane (Greenway and Dyke, 1979). 2. Short-Chain Organic Acids
The mode of entry of SCFA into the microbial cell has not been established, although it is assumed that only the neutral (undissociated) acid molecule enters the cell (Baskett and Hentges, 1973; Cramer and Prestgard, 1977; Chu et al., 1987). Since the acid molecules are lipid soluble, it has been presumed that they diffuse freely across the cell membrane (Salmond et al. , 1984; Nunn, 1987). However, a number of reports suggest that energylinked carriers and the membrane potential (described in Section III.D.2) may be involved in their uptake, e.g. acetoacetyl-CoA transferase and ATP are required for E. coli to grow on butyrate (Vanderwinkel et al., 1968) and for butyrate and acetate uptake in Clostridium acetobutylicum (Wiesenborn et al., 1989). Salmonella spp. transport citrate via a specific permease (Sommers et al., 1981) while, in methylotrophic bacteria, a pH difference (ApH) across the cell membrane is required to accumulate formic acid (Chu et al., 1987). Bacteria can utilize acetic acid as a carbon and energy source by inducing enzymes of the glyoxylate pathway, isocitrate lyase and malate synthase, which allows net assimilation of carbon (Nunn, 1987). Escherichia coli is also able to utilize propionic acid as a sole carbon and energy source (Wegener et al., 1968) and, although Kay (1972) identified two routes for propionate metabolism, a-oxidation to pyruvate and metabolism via hydroxyglutarate, the precise pathway is not known (Nunn, 1987). Growth in the presence of calcium propionate results in synthesis of fatty acids of unusual composition, namely saturated CI5and CI7and unsaturated CI7fatty acids, probably as a result of using propionyl-ACP as a primer for fatty acid synthesis instead of acetyl-ACP (Ingram, 1977).
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C. A. CHERRINGTON ET Al..
C. GENERAL OBSERVATIONS
The following section considers published reports which deal with the effects of organic acids on microbial growth. For reviews of specific acids see Bosund (1962; benzoic, salicylic), Sofos and Busta (1981; sorbic) and Sofos et al. (1986; sorbic), while for a general review of the mode of action of various compounds used as preservatives and antiseptics see Freese and Levine (1978). Organic acids are more effective than mineral acids as antimicrobial agents (e.g. Nunheimer and Fabian, 1940; Reynolds, 1975) and, although they exhibit broad-spectrum antimicrobial activity, the antibacterial efficiency of individual acids varies (Nunheimer and Fabian, 1940; Cowles, 1941; Barker, 1964; Goepfert and Hicks, 1969; Minor and Marth, 1972; Sheu et al., 1975). For instance, in a study involving Staphylococcus aureus, Minor and Marth (1972) found that acetic and lactic acids were more active than hydrochloric acid and, while mixtures of hydrochloric and lactic acids were superior to either alone, there was no additive effect when acetic and hydrochloric acids were mixed together. Vegetative cells are more sensitive to organic acids than the corresponding spore forms (Wong and Chen, 1988) with bacteria showing increasing sensitivity to acids as concentration and (with the exception of Gramnegative species) chain length increase (Reid, 1932; Cowles, 1941; Barker, 1964; Freese et al., 1973; Sheu et al., 1975; Eklund, 1980; Brackett, 1987). Although Gram-negative bacteria can actively transport these acids into the cell and metabolize them via the P-oxidation cycle, Sheu and Freese (1973) showed that resistance was caused by the lipopolysaccharide layer in the cell wall, which prevents the acids from entering the cell. The antimicrobial activity of organic acids increases with decreasing pH value (Cohen and Clarke, 1919; Bosund, 1962; Hentges, 1967; Freese etal., 1973) and, since a greater proportion of undissociated molecules exist as the pH value decreases (see Section II.B), it has been assumed that it is the undissociated molecule that is the antimicrobial agent (e.g. Reid, 1932; Bergeim, 1940; Cowles, 1941; Barker, 1964). However, this takes no account of the behaviour of the acid inside the cell. The pH value of the cytoplasm of bacteria is thought to be regulated strictly, with estimates ranging from 7.4 to 7.6 (Padan et al., 1981; Slonczewski etal., 1981) and 8.2 to 8.7 (Lagarde, 1977; Booth etal., 1979) over an external pH range of 5.59. This means that once the acid molecules have entered the cell (Freese et a l . , 1973; Salmorid et al., 1984) they will dissociate almost totally, assuming that dissociation of the acid in cytoplasm is the same as in aqueous solutions, and it is probable that the proton and anion both contribute to inhibition of growth of the bacteria (Rubin et al., 1982; Eklund, 1983,1985; Salmond ec al., 1984; Cherrington, et al., 1990a). Indeed, Eklund (l983,1985),’using a
ORGANIC ACIDS
95
mathematical model, calculated that the anions of benzoic, propionic and sorbic acids contributed to more than 50% of the growth inhibition of E. cofi in a medium of pH 6 or more. D.
THE CELL MEMBRANE
The cell membrane has been identified as one of the potential sites of action of organic acids. Other “targets” include enzymes (Section III.E), macromolecule synthesis (Section 1II.F) and DNA (Section 1II.G). I . Cell-Membrane Integrity
Organic acids have been regarded as anionic surfactants acting as membrane-disrupting disinfectants (Sheu and Freese, 1972; Kondo and Kanai, 1976; Greenway and Dyke, 1979). However, cell lysis has only been reported with LCFA where the protective cell wall has been removed, e.g. protoplasts of Bacillus megaterium and Micrococcus lysodeikticus (Galbraith and Miller, 1973) and membrane vesicles of Bacillus subtilis (Freese et al., 1973). In contrast, LCFA did not induce lysis in the cell-walllacking mycobacteria (Kondo and Kanai, 1976) and 0.5 M formic or propionic acids, buffered to pH 5, did not disrupt E. coli cell membranes (Cherrington et al., 1991a). Statham and McMeekin (1988) showed that the addition of lyzozyme was required to produce lysis in Alteromonas putrefaciens treated with sorbate while Tsuchido et al. (1985) proposed that dodecanoic acid induced autolytic enzymes in B. subtilis. Interference with membrane permeability, characterized by leakage of cellular proteins or ions (e.g. K’, Na+) has been reported for Gram-positive bacteria and mycobacteria incubated with LCFA (Galbraith and Miller, 1973: Kondo and Kanai, 1976; Greenway and Dyke, 1979) while Corlett and Brown (1980) suggested that weak lipophilic acids cause leakage of protons across the cell membrane, which then acidify the cytoplasm and inhibit nutrient transport. On the other hand, acetate buffer at pH 4.2 did not cause leakage of protein in E. colicultures (Przybylski and Witter, 1979) and at pH 3.2-3.5 produced only slight leakage in Salmonella bareilly cultures (Blankenship 1981). 2. Cell Membrane Functions
Minor disturbances of the cytoplasmic membrane which inactivate energylinked reactions can prevent growth. The selective permeability of the cytoplasmic membrane produces an ionic gradient (which drives the transport of certain substrates) and is involved in energy generation and
96
C . A. CHERRINGTON ET A L
regulation of the internal pH value (for a review, see Booth, 1985). This electrochemical proton gradient ApH+ (also known as the proton-motive I a proton gradient ApH force, or Ap) comprises an electrical gradient A ~ and as follows:
A p = A w - ZApH where Z = 2.3(RT/F)with R the gas constant in J K-’, Tthe thermodynamic temperature and F the Faraday constant In general, as external pH value decreases, Ap is maintained by increasing A ~toI compensate for the increase in ApH (Ramos and Kaback, 1977; Padan et al., 1981). The general mechanism of microbial growth inhibition by organic acids was considered to be the acidification of the cell cytoplasm as a result of the release of excess protons following dissociation of the acid (Baird-Parker, 1980). Salmond et al. (1984) compared the effect of acid concentration on cytoplasmic pH (internal pH or pHi) and demonstrated a lowering of the pHi and a reduced growth rate as acid concentration increased. However, it was concluded that the drop in pHi alone was unlikely to be the primary cause of growth inhibition, and a synergistic effect of H+ and accumulated undissociated acid was suggested. By definition, acidification of the cytoplasm would reduce the ApH and, consequently, organic acids have been termed “uncoupling agents” because they rapidly shuttle protons across the membrane, thereby dissipating the proton-motive force (Finkelstein, 1970; Levin and Freese, 1977; Freese and Levin, 1978; Baronofsky et al., 1984; Herrero et al., 1985). This claim is supported by reports that Ap-driven substrate-transport systems are inhibited in the presence of organic acids (Freese et al., 1973; Sheu et al., 1975; Eklund, 1980). However, Eklund (1985) showed that sorbic acid abolished ApH, but not Ay, and since A ~ provides I a sufficient energy gradient on its own to “drive” the uptake of several substances in E. coli (e.g. cysteine, leucine, proline and succinate; Ramos and Kaback, 1977) concluded that inhibition of substrate transport was not the primary target of organic acids. ATP levels in B. subtilis and E. coli are decreased following incubation with organic acids, although this is not a primary cause of growth inhibition (Sheu and Freese, 1972; Freese et al., 1973; Sheu et al., 1975; Cherrington, 1990). In addition, although organic acids inhibit oxidative metabolism in E. coli and B. subtilis, the concentration of acid needed to inhibit the process by 50% was higher than that required to inhibit bacterial growth (Weiner and Draskoczy , 1961).
ORGANIC ACIDS
97
E. ENZYME ACTIVITY
In general, the activity of enzymes is lowered by acid pH values, and this may be a secondary effect of the acidification of the cytoplasm. Octanoic acid selectively inhibits glucose-phosphate dehydrogenase, pyruvate kinase, fumarase and lactate dehydrogenase of Arthrobacter crystallopoietes (Ferdinandus and Clarke, 1969), although this may reflect the specific sensitivity of these enzymes to p H value. However, York and Vaughn (1964) reported that inhibition of the sulphydryl enzymes fumarase, aspartase and succinate dehydrogenase by sorbic acid was the result of the acid reacting with the thiol group of cysteine. Studies involving the yeast Cundida utilis showed that the respiratory rate and extracellular concentration of unused acetate were influenced by the pH value of the culture medium, with the toxicity of the acetate being due to the uncoupling of oxidative phosphorylation (Hueting and Tempest, 1977) while the inhibitory activity of organic acids for Thiobacillus ferrooxidans was due to the direct inhibition of the iron-oxidase system (Tuttle and Dugan, 1976). F. MACROMOLECULE SYNTHESIS
The sensitivity of individual biosynthetic functions to acids appears to vary depending on the bacterium and the acid. For example, potassium sorbate inhibits protein, RNA and DNA syntheses equally in Pseudomonas fruorescens (Nose, 1982), while protein synthesis is primarily inhibited by azelaic acid in Staphylococcus epidermidis (Bojar et al., 1988). Both formic and propionic acids were most active in vitro against DNA synthesis in E. coli, although lipid, peptidoglycan, protein and RNA syntheses were also inhibited (Cherrington et al., 1990a). Inhibition of macromolecule synthesis may reflect the sensitivity of the biosynthetic enzymes to acidification of the cytoplasm. However, Cherrington (1991a,b) suggested that the cytoplasmic pH value recovered in the presence of sublethal concentrations of formic acid in the absence of a corresponding recovery in the rate of DNA synthesis, although rates of lipid, peptidoglycan, protein and RNA syntheses showed some recovery. G . DNA
Sinha (1986) incubated stationary-phase DNA-repair-deficient mutants of E. coli with acetic and lactic acids (pH 3.5) and reported an increased sensitivity of the polA2 mutant (which lacked DNA polymerase activity) compared to its isogenic parent, implying that the acids caused physical damage to the DNA molecule. Stephens and Dalton (1987) also found that
98
C A CHERRINGTON ET A L
acids can damage DNA because benzoate, and to a lesser extent acetate and butyrate, induced both base deletions in To1 plasmids and plasmid curing in Pseudomonas putida, possibly by excision of a transposon-like region. In contrast, Cherrington et al. (1991b) were unable to detect an increased sensitivity of log-phase cultures of polAZ, recA56 or uvrA6 mutants to formic and propionic acids (pH 5) compared to their isogenic parent strain. Similarly, there was no evidence of an SOS response in E. coli, in which damage to the DNA molecule is manifested by the cell forming filaments (Little and Mount, 1982) after exposure to sublethal concentrations of the same acids. Cherrington (1990) suggested that the acid anion may interfere with the conformation of the DNA molecule by interacting with ion charges around it. The differences in anion structure could then explain the differences in activity of organic acids with the same, or similar, pKa values, e.g. lactic acid (pK, 3.1) is more active than citric acid (pK, 3.1), sorbic acid (pK, 4.8) is more active than propionic acid (pKa 4.9), and cinnamic acid (pKa 4.4) is more active than benzoic acid (pK, 4.2) (Sheu et al., 1975; Salmond et al., 1984; M. Hinton, unpublished observations). H. RECOVERY FROM AND RESISTANCE TO INHIBITION
Inhibition of organic acids appears reversible, since acid-treated cells will grow when placed in an acid-free medium (Przybylski and Witter, 1979; Blankenship, 1981), with recovery of Salmonella bareilly (Blankenship, 1981) and Sfaphy1ococcu.saureu.s (Zayaitz and Ledford, 1985) from acetic acid injury requiring both protein and RNA synthesis and an active respiratory (electron-transport) chain. Resistance of bacteria to organic acids has not been reported, although E. coli can adapt to utilize propionic acid (Wegener et al., 1968), acetic acid and LCFA (reviewed by Nunn, 1987) as sole carbon and energy sources. IV. Practical Applications
This section of the review will concentrate principally on two aspects of the use of organic acids: firstly, in animal husbandry as animal feed additives and, secondly, in abattoirs and food-processing plants where they may be used in controlling microbial contamination of carcass meat. The section will conclude with brief reference to their use in other human foods and in other products destined for human use.
ORGANIC ACIDS
99
A. ANIMAL-FEED ADDITIVES
Silage is a feed material produced by the fermentation of high-moisture crops such as grass, legumes and maize. In order to produce a high-quality product it is essential that pH values of these crops are lowered as rapidly as possible after harvesting, and this process can be hastened by the addition of both inorganic and organic acids (Woolford, 1984) with formic acid being commonly used as either the sole ingredient or in combination with other chemicals. Woolford (1975) evaluated the straight-chain acids up to CI2and concluded that all had potential for use as silage additives. Formic and propionic acids were useful in aiding the production of conventional fermented silages; the higher acids, which are likely to be too expensive for commercial use, produced a silage which was not fermented, while some acids, e.g. butyric, valeric and caproic acids, would prove unacceptable because of their unpleasant smell. Organic acids have been added to animal feed as an alternative to heat treatment to control microbial contaminants (Francis and Turnbull, 1979), although they will not necessarily eliminate pathogens such as the salmonellas (Kahn and Katamay, 1969; Duncan and Adams, 1972;Vanderwal, 1979; Banton et al., 1984; Humphrey and Lanning, 1988). The efficiency of these agents as disinfectants can be increased by increasing their concentration (Vanderwal, 1979; van Staden et al., 1980; Rejholec, 1981). This approach may be justified when heavily contaminated ingredients are being used, although this may reduce palatability (Rys and Koreleski, 1974;Cave, 1984) unless the treated ingredient is diluted by subsequent mixing with other matrials. The addition of formic acid to the feed of laying hens lowered the incidence of salmonella infections in their newly hatched progeny, although this benefit did not carry through to slaughter because the chicks were given untreated feed (Humphrey and Lanning, 1988). Similarly, Hinton and Linton (1988) reported that a mixture of formic and propionic acids, which can be expected to have no ultimate effect on the organoleptic quality of the carcass meat (Basker and Klinger, 1979), effectively reduced salmonella colonization in chicks given contaminated acid-treated feed. However, it was still possible to isolate salmonellas from the acid-treated feed itself (Hinton and Linton, 1988), which suggests that the acids may exert their effect in the birds’ crops after the feed has been moistened following consumption. It is also necessary to give acid-treated feed throughout the rearing period, since the acids have no beneficial effect once the birds have become infected (Hinton and Linton, 1988). The acidification of water has also been proposed as a way of controlling salmonella infections. This procedure could reduce water consumption by young chicks since they appear less tolerant of water with acid pH values
100
C. A. CHERRINGTON ET AL
than are older birds (Fuerst and Kare, 1962), but may prove effective in reducing cross-infection via contaminated water. However, it was of no value in controlling feed-borne infections (Al-Chalaby et al., 1985). Acidified milk replacers are used to feed unweaned calves, and their use has been associated with a reduction in coliform numbers in milk prepared on the farm (Morgan-Jones and Hinks, 1980) and in the faeces of young calves fed these products (Simm et al., 1980; Humphrey er al., 1982). However, there is little evidence to suggest that they have any beneficial effect either nutritionally or for the control of enteric disease caused by E. cofi (Stobo, 1983). The acidification of the diets of young pigs has been reviewed by Easter (1988). The principal reason for incorporating acids (e.g. citric or lactic acid) in their feed was to minimize the check in growth rate which invariably occurs after weaning and is frequently associated with the development of diarrhoea. There is a substantial literature which indicates that pigs respond well to an acidified diet during the weeks following weaning, and Easter (1988) concluded that the greatest benefit occurred when the diets comprised cereal grains and plant proteins and contained no lactose, although Burnell et a f . (1988) have recorded improved performance with diets containing whey powder. B. TREATMENT OF CARCASS MEAT AND EGGS
Organic acids, especially acetic and lactic acids, have been widely studied as potential decontaminating agents for red meat, principally from cattle and sheep, and poultry carcasses. Most studies concerning red meat have involved either dipping the meat in a treatment solution, which requires frequent renewal to maintain activity (Eustace, 1984), or the use of spray systems that deliver a single or repeated application. In a study of lamb carcasses that involved spraying or dipping (Anderson et al., 1988) the meat was treated with 1.5 or 3.0% acetic acid at 25 or 55°C. Each treatment reduced counts significantly in comparison with untreated controls, with dipping in 3% acid at 55°C being the most effective. Hamby et al. (1987) treated beef carcasses by intermittent spray chilling using water, 1%acetic or lactic acid, or a single spray treatment with either acid. Intermittent spraying gave the best results, lowering total viable counts from portions of meat by 100,000-fold following storage of the meat in vacuum packs at 2°C for 28 days. Reductions observed 48 hours after treatment are shown in Table 4. For beef artificially contaminated with faecal bacteria and then dipped for 15 seconds in acetic acid at concentrations ranging from 1 to 3% and temperatures varying from 25 to 70°C, treatment with 3% acid at 70°C was
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ORGANIC ACIDS
TABLE 4. Mean total viable counts (log,&m2) from five sampling sites on beef sides receiving intermittent spraying with water or organic acid Sampling site
Water
Acetic acid
Lactic acid
Inside round
Control Treated Difference
2.9" 2.5" 0.4
2.3" 1 .5" 0.8
2.6" 1 .Sb 0.8
Strip loin
Control Treated Difference
2.2" 2.4" -0.2
3.4"
1.4"
2.0
2.6" 1 .s6 1.1
Boneless rib
Control Treated Diffefence
2.6" 3.8' -1.2
4.5" 2.16 2.4
3.4" 1.36 2.1
Clod
Control Treated Difference
3.0" 3.2" -0.2
2.8' 1.16 1.7
3.2" 1.26 2.0
Inside neck
Control Treated Difference
2.5" 2.1"
2.6" 1.8" 0.8
3.Y
0.4
1.56
2.4
Means represent data from four carcasses. Means within same sampling site and treatment with different superscripts differ significantly (P
