Traditional Preservatives – Organic Acids JB Gurtler, US Department of Agriculture, Wyndmoor, PA, USA TL Mai, IEH Laboratories and Consulting Group, Lake Forest Park, WA, USA Ó 2014 Elsevier Ltd. All rights reserved. This article is a revision of the previous edition article by M. Stratford, volume 3, pp. 1729–1737, Ó 1999, Elsevier Ltd.
Introduction
grape-based beverages has become a common industrial practice due to the stabilizing effect of these organic acids and the resulting ability to enhance product shelf life. The antimicrobial and corresponding health-promoting nature of organic acids has been known for thousands of years. In geographic regions where water quality is routinely unsanitary, common cultural practices have been adopted whereby small quantities of lemon juice or wine are added to the drinking water prior to potation. This process of acidifying beverages to pH levels less than 4.0 is known to inactivate harmful bacteria or sensitize them to subsequent inactivation in the gastrointestinal tract when consumed immediately thereafter. For example, allusion to this ancient practice is made in the New Testament scriptures, where the Apostle Paul directs the young evangelist Timothy, “No longer drink only water, but use a little wine for your stomach’s sake and your frequent infirmities” (1 Tim. 5:23, New King James Version), a statement that is reputed by many to be in reference to the purification effect achieved by diluting water with wine. Antecedent to this, it is recorded by Titus Livy that the great military commander Hannibal and his Carthaginian army carried substantial quantities of sour wine for this very purpose while crossing the Alps into Northern Italy during the Second Punic War. In fact, the word vinegar is derived from the Latin words ‘Vinum’ and ‘Egre’ (Vinegre), which literally translates to ‘wine sour’ or spoiled wine. Use of sour wine during this period of history was one of the only reliable ways of ensuring uncontaminated water, especially when an army was on the march. The Roman armies were also known to be supplied amply with sour (acetified) wine, purportedly for this very purpose, as well as
There is a common misperception that the addition of acids to foods is problematic due to the corrosive and toxic properties of this class of compounds. This perspective is countered by the reality that acids that are derived from organic compounds or organic acids are ubiquitous substances found throughout nature that participate in the most vital biochemical pathways in the human body (e.g., pyruvic acid and citric acid). Furthermore, the addition of organic acids to foods and beverages is one of the most ancient practices, providing an extremely simple and effective method for preventing foodborne illness and preserving food products. The most common foodborne pathogenic bacteria of interest are unable to grow at pH levels lower than pH 4.0 (Figure 1), although the most common microorganisms capable of growing in foods at pH levels lower than 4.0 are typically yeasts, molds, and some acidophilic spoilage bacteria, which serve as bioindicators of unwholesomeness, including Alycyclobacillus, Lactobacillus, Lactococcus, Leuconostoc, and Bifidobacterium. In addition to food safety aspects, the addition of organic acids to foods also imparts flavor and antioxidant activity and maintains organoleptic properties over extended shelf-life periods. Organic acids have played a significant role in foods and beverages for thousands of years. Tartaric acid residues have been found in Iranian wine jars, which some believe to date from 5000 to 5400 BC. The natural acids from grapes, including tartaric and citric acids, enhance the flavor and add to the antioxidant properties of grape food products and beverages. The addition of citric, malic, or tartaric acids to
Salmonella Campylobacter Escherichia coli Staphylococcus aureus Listeria Bacillus cereus Clostridium botulinum Lactobacillus Acetobacter Yeasts Molds 2
3
4
5
6
7
pH range for growth Figure 1 Typical minimum pH levels necessary for the growth of pathogenic bacteria commonly involved in foodborne illnesses, as well as for lactic acid- and acetic-acid-producing bacteria, yeasts, and molds. Low pH levels restrict the growth of pathogens but allow the growth of spoilage yeasts, molds, and bacterial acidophiles.
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due to its lower expense in comparison to the more costly nonacetified fermented alcoholic wines.
Organic Acid–Based Acidification of Foods The range of foods containing organic acids is extensive and includes such products as colas, sport drinks, fruit juices and fruit products, spreads, sauces, dressings, mayonnaise, pickles, pickled eggs and meats, salsa, sauerkraut, kimchi, tea, coffee, cocoa, fermented sausage, vinegar, kombucha, candies, desserts, soy sauce, canned vegetables, yogurt, and various other fermented dairy products. Some foods are manufactured to include the artificial addition of acids, while other acids are intrinsic to the food itself or are generated by microbial agents in the food during the fermentation process. Organic acids are entirely natural components of many foods, notably fresh fruits and fruit juices. Grape juices and wine characteristically contain tartaric acid. Malic acid is found in apples, and citric acid is found in citrus fruits, such as lemons, grapefruit, and oranges (see Table 1 for a list). Many fruit juices contain a mixture of organic acids, with citric and malic acids being most commonly found in substantial quantities. Acids commonly added to foods are not generally chemically pure compounds, rather, they are incorporated as subcomponents of natural substances that are added to foods during the manufacturing process. The concentrations of acids in fruit juices obtained from ripened fruits are approximately 1%, although black currant can contain up to 4% citric acid, which may contribute partially to the unusually high antioxidant content of black currant in comparison to other fruits. Acid levels in fruits vary considerably with ripeness: Unripe fruits contain a higher concentration of acid but little sugar, in fact, unripe lemons may contain as much as 5–8% acid content. Inversely, overripened fruits
Table 1
contain significant sugars but minimal acid content. The legislation concerning food additives varies widely from country to country. As such, organic acids may be categorized into one of several food additive groups including: Acidulants – acids added to increase the acidity of a food and/or to impart a sour taste l Flavors – acids added for artificial flavoring l Antioxidants – acids that preferentially combine with oxygen compounds, thus preventing deterioration of the food by oxygen-free radicals or other oxygenated reactive species l Preservatives – acids that protect foods against deterioration caused by microorganisms l
Since most acids exhibit a variety of chemical properties, it is possible for a given acid to be classified into several categories. For example, the addition of acetic acid to a food item can increase the acidity of the food, impart a distinctive flavor, and act as a preservative. In the European Community, however, regulations on this classification system are more restrictive, where sorbic acid, benzoic acid, and propionic acid are all listed as preservatives (described fully elsewhere); ascorbic acid is considered an antioxidant; and citric, malic, lactic, tartaric, and acetic acids are recognized solely as acidulants. In the United States, by comparison, food additives, including many organic acids, are recorded on a Food and Drug Administration (FDA)– approved list termed generally regarded as safe (GRAS) compounds, enabling premarket clearance from the FDA, without the need for further classification. Acids that have been approved GRAS by the US FDA for specified purposes in foods include acetic, ascorbic, benzoic, butyric, caprylic, citric, formic, lactic, malic, propionic, sorbic, succinic, and tartaric acids, in addition to some of their salts (e.g., calcium acetate, sodium acetate, calcium ascorbate,
Range of pH values and major acids present in various fruits, at a normal ripeness
Fruit
pH range
Major acids
Other acids
Apple Blueberries Cherry Cranberry Grape Grapefruit Guava Kiwifruit Lemon Lime Lingonberry Mango Orange Papaya Passion fruit Peach Pear Pineapple Plum Red raspberry Strawberry Tomato
2.9–4.5 2.8–3.2 3.7–4.4 2.2–2.5 2.9–3.9 2.9–3.6 3.2–4.2 3.1–4.0 2.0–2.6 1.6–3.2 2.6–2.9 4.3–6.0 2.6–4.3 5.2–5.7 2.6–3.4 3.6–4.0 3.0–4.5 3.1–4.0 3.0–4.5 2.5–3.3 3.0–3.5 4.1–4.7
Citric, malic Quinic, citric Ascorbic, citric Malic, citric Malic, tartaric Citric, quinic Citric, malic Quinic, citric Citric, quinic Citric, quinic Citric, malic Citric, tartaric Citric, quinic Citric, malic Citric, malic Malic, citric Malic, citric Citric, malic Malic, quinic Citric, malic Citric, ascorbic Citric, ascorbic
Quinic, tartaric, caffeic, ferulic, benzoic Malic, ellagic, chlorogenic, salicylic Malic, tartaric, quinic, shikimik Benzoic, quinic, ellagic, oxalic Quinic, ellagic, citric Malic, tartaric, oxalic, succinic, ascorbic, ferulic, dehydroascorbic Ellagic, salycilic Malic, oxalic, ascorbic, Malic, tartaric, oxalic, succinic, ascorbic, ferulic, dehydroascorbic Malic, tartaric, oxalic, succinic, ascorbic, ferulic, dehydroascorbic Benzoic, salycilic, lactic Anacardic, gallic, dehydroascorbic, ascorbic, malic Malic, tartaric, oxalic, succinic, ascorbic, ferulic, dehydroascorbic Dehydroascorbic, ascorbic, oxalic, tartaric, quinic, succinic, fumaric Ascorbic, dehydroascorbic, nicotinic, Tartaric, chlorogenic Caffeic, quinic, tartaric, fumaric, shikimik, lactic, succinic, oxalic, acetic Quinic, tartaric, chlorogenic, ferulic, oxalic Citric, fumaric, benzoic Isocitric, hydroxybenzoic, benzoic Malic, tartaric, hydroxybenzoic, ellagic, gallic, chlorogenic Oxalic, salycilic, ascorbic, malic, glutamic, aspartic
PRESERVATIVES j Traditional Preservatives – Organic Acids sodium ascorbate, sodium benzoate, calcium citrate, calcium diacetate, manganese citrate, potassium citrate, sodium citrate, calcium lactate, calcium propionate, sodium propionate, calcium sorbate, potassium sorbate, sodium sorbate, and sodium tartrate). The amount of acids added to foods depends on the type of acid, the food substance, the desired organoleptic properties, and the specific purpose for which the acid is added. For example, acidulants generally are added in large quantities (several parts per 100), whereas preservatives, flavors, and antioxidants are added more sparingly (e.g., 100–500 parts per million).
Citric Acid Citric acid is one of the most versatile, inexpensive, and widely used organic acidulants, and it commonly is applied to the production of fruit-flavored beverages. It is contained in all fruits listed in Table 1 and represents one of two major acid constituents contained in most of these fruits. In addition, citric acid is also used in jams, confectioneries, candy, cheeses, juices, wine, canned vegetables, and sauces. Owing to its widespread usage, citric acid has become the gold standard against which other acidulants are measured, including such parameters as taste, titratable acidity, and acidification. In particular, citric acid is highly favored by the food industry on account of its light fruity taste, solubility, low cost, and abundant supply.
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Furthermore, when dissolved in hard water, undesirable insoluble precipitates of calcium tartrate can form.
Acetic Acid and Vinegar Because of its pungent odor and taste, acetic acid is used in substantial amounts but it is somewhat limited in food applications for products such as pickles, chutney, salad creams, mayonnaise, dressings, and sauces. Nevertheless, it is often the acid of choice in these foods precisely because of its organoleptic properties and it almost always is applied to foods in the form of vinegar. Acetic acid occurs naturally in trace amounts in some fruits, such as pears. Vinegars typically contain between 4 and 8% acetic acid and are formed by the action of acetic acid bacteria on ciders, wines, or yeastfermented malt. Vinegars often are sold in the form of white (distilled), apple cider, malt, wine, sherry, Balsamic, rice, coconut, palm, cane, raisin, date, beer, honey, East Asian Black, Job’s tears, Kombucha, Kiwifruit, sinamak, and spirit vinegars. Balsamic vinegars are highly prized, of Italian origin, having been made in the Modeno Reggio Emilia and as far back as 1046 BC. Authentic Balsamic vinegar is made from grape syrup having been aged for 12–25 years in a succession of barrels composed of chestnut, acacia, cherry, oak, mulberry, ash, or juniper wood. Prime Balsamic vinegars can sell for more than US$11 000 per gallon (or US$3000 per liter).
Lactic Acid Malic Acid Malic acid, like citric acid, is a general-purpose acidulant. It normally is associated with apples; in fact, its common name is derived from the Latin word for apple, malum, although it is also a major acid constituent of cranberries, grapes, guava, lingonberries, papaya, passion fruit, peaches, pears, pineapple, plums, and raspberries (Table 1). Although it is used in many food products, it often is preferred in apple-containing foods, such as ciders, due to its flavor and relatively higher cost when compared with citric acid. Malic acid, however, has a fuller, smoother taste than citric acid that is beneficial in low-energy drinks, where malic acid masks the unpleasant flavors of some artificial sweeteners. It is positioned economically between citric and tartartic acids in price.
Tartaric Acid Tartaric acid has a stronger, sharper taste than citric acid. Although it is renowned for its natural occurrence in grapes, it also occurs in apples, cherries, papaya, peach, pear, pineapple, strawberries, mangos, and citrus fruits. Tartaric acid is used preferentially in foods containing cranberries or grapes, notably wines, jellies, and confectioneries. Commercially, tartaric acid is prepared from the waste products of the wine industry and is more expensive than most acidulants, including citric and malic acids. Tartaric acid is one of the least antimicrobial of the organic acids known to inactivate fewer microorganisms and inhibit less microbial growth in comparison with most other organic acids (including acetic, ascorbic, benzoic, citric, formic, fumaric, lactic, levulinic, malic, and propionic acids) in the published scientific literature.
Lactic acid has a very smooth, mild taste compared with other acidulants. It naturally occurs in trace amounts in some fruits, including lingonberries and pears, and is one of the principal organic acids reputed for its antimicrobial activity, especially in fermented foods. Lactic acid is used at substantial concentrations in fermented meats, dairy products, sauces, brinepreserved pickled vegetables, and salad dressings. It is also used in carbonated beverages, as a flavor modifier, as well as in fruit and vegetable preserves.
Phosphoric Acid (Inorganic Acid) Phosphoric acid, although an inorganic acid, is worthy of mention in this chapter. It is used predominantly as an acidulant, almost exclusively in the production of carbonated beverages, although its use in foods bears controversy due to its effects on health. Comparatively, phosphoric acid is extremely inexpensive, possessing a characteristic flat sour taste that is reminiscent of citric acid. It is a relatively strong, dissociated acid, enabling it to easily acidify colas to the low desired pH (2.5) needed to establish proper carbonation, although its antimicrobial efficacy is far inferior to most organic acids, principally due to its dissociated state, which precludes ease of transport across the bacterial membrane.
Fumaric Acid Fumaric acid is an acidulant that possesses a fruitlike flavor. It occurs naturally, albeit in limited amounts, in such fruits as papayas, pears, and plums. Fumaric acid has FDA GRAS status in the United States, but its application is not permitted in
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Europe. In the United States, fumaric acid is used principally in fruit juices, gelatin desserts, tortillas, and pie fillings. It is relatively cheap, but it has the great disadvantage of a stronger taste than citric acid and is difficult to dissolve in water. The solubility of fumaric acid, in fact, is only w6 g l1 (i.e., 0.6%), which is further complicated by the extended times necessary for solubility concentrations to go into solution. For this reason, solubility often is hastened by heating the solvent, which frequently precludes its use for many food industry applications.
Adipic Acid Adipic acid is rarely encountered in the United States, although it occasionally is used as an acidulant of fruit-flavored beverages, jellies, jams, and gelatin desserts. Additionally, it is used in the formulation of antacids on account of its characteristic tart flavor. It is favored in dry foods because it is not hygroscopic, thus not absorbing moisture from ambient air.
Levulinic Acid Recent work out of the University of Georgia, which led to a subsequent patent and product commercialization, combined various concentrations of levulinic acid and sodium lauryl sulfate (sodium dodecyl sulfate (SDS)) resulting in synergistic bacterial inactivation. Although levulinic acid is less well known than other organic acids, numerous recent research publications touting the efficacy of levulinic acid plus SDS for pathogen inactivation on feathered poultry carcasses, chicken wings, poultry processing water, fresh sprouts, lettuce, alfalfa seeds, pecans, chicken cages, food-processing contact surfaces, and biofilms shows promise for industrial application.
Ascorbic Acid Ascorbic acid and its associated salts are used frequently as an antioxidant in canning and to prevent browning in cut fruit and vegetables and have GRAS status.
Benzoic Acid Benzoic acid primarily is precluded from use in foods due to its extremely low solubility, although its associated salts are used more frequently. For example, sodium benzoate is FDA GRAS for use as a preservative in foods, primarily to prevent the growth of yeast and molds, especially in food products with a pH of less than 4.5.
Cinnamic Acid Cinnamic acid is a common constituent in numerous plants, although it is produced synthetically. It is known to have a strong broad-spectrum antimicrobial effect, but it is precluded for use in most foods due to its strong organoleptic properties.
Formic Acid Formic acid is known for its strong antimicrobial effects, although it is used principally as an antimicrobial in livestock feeds and forages. Formic acid (HCOOH) has the shortest
chain length of any organic acid, which may contribute its antimicrobial properties.
Toxicity Toxicity of most organic acids, as determined by oral ingestion in animal models, is generally low, ranging from LD50 values (i.e., a dose necessary to induce mortality in 50% of the test population of animals) of 1000 mg kg1 of body weight for dehydroacetic acid up to 11 700 mg kg1 of body weight for citric acid, both as determined using a rat model. When administered intravenously, the LD50 is lower and ranges from 42 mg kg1 of body weight for citric acid in a mouse model up to 2430 mg kg1 of body weight for adipic in a rabbit model. The Food and Agriculture Organization (FAO) of the United Nations has set no limit on the daily intake of the following organic acids in the human diet: acetic, citric, lactic, malic, and propionic, whereas other acids have set limits (e.g., fumaric and tartaric at a maximum daily intake of 6 and 30 mg kg1 of body weight, respectively).
Behavior of Various Organic Acids in Foods Chemical Properties of Organic Acid Acidulants The most common types of organic acids are the carboxylic acids, a subclass of acids possessing carboxyl groups. Carboxylic acids vary in the number of acidic groups present on each molecule. For example, citric acid and isocitric acid are tricarboxylic acids possessing three dissociation constants (i.e., pK1, pK2, and pK3); ascorbic, malic, tartaric, fumaric, succinic, and adipic acids are dicarboxylic acids, possessing two dissociation constants (i.e., pK1 and pK2); and lactic, acetic, benzoic, butyric, cinnamic, formic, gallic, propionic, pyruvic, and sorbic acids are all monocarboxylic possessing only one dissociation constant (i.e., pK1). Acidification by acids requires the release of protons (Hþ) from the molecule. The attributed strength of an acid is a function of the ability of an acid to release a proton. Fully dissociated, strong acids, such as hydrochloric acid (HCl), effectively release all protons at the pH range of foods. In contrast, acidulants of foods are described as weak acids, exhibiting only partial dissociation in typical pH ranges. Weak acids in solution form an equilibria between an undissociated state and charged anions and protons as follows: HA ðundissociated acidÞ % A ðanionÞ þ Hþ ðprotonÞ: Equilibria are pH-dependent. At lower pH values, the proton concentration is higher, pushing the equilibrium toward a more undissociated acid. Figure 2 shows the dissociation curve for acetic acid. At a pH below 3.0, acetic acid exists almost entirely as an undissociated molecular acid, whereas above pH 6.5, it is almost entirely dissociated into acetate anions. The pKa value is considered the pH at which the acid and anion coexist in equal proportions. Weak acids in solution form buffers that resist changes in pH. Maximal buffering capacity occurs at the pKa value (see Figure 2) with the effective buffering range extending 1 pH unit on either side of the pKa. Food acidulants have a variety of pKa values (Table 2) and thus are dissociated to different extents at any given pH. Some
% Undissociated acid /buffering capacity (units)
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100 pK a value
80 60 40 20 0
2.25 2.75
3.25
3.75
4.25
4.75
5.25
5.75
6.25
6.75
7.25
pH value Figure 2 The effect of pH on the proportions of undissociated acetic acid and acetate anions. At the pKa value, acid and anion are present in equal proportion. Buffering capacity of acetic acid (histograms) was determined by titration. One unit of buffering capacity is the proton concentration (mmol l1) required to move the pH of 1 l by one unit.
Table 2 Acid
Chemical properties and structure of commonly used food acidulants Structure
Citric
COOH
Mol. wt.
pKa
log poct
192.13
5.7; 4.3; 2.9
1.222
134.09
4.7; 3.2
1.984
150.09
3.9; 2.8
2.77
116.07
4.0; 2.8
0.748
90.08
3.66
0.186
60.05
4.7
0.168
HOOCCH2CCH2COOH OH Malic
HOOCCHCH2COOH OH
Tartaric
OH HOOCCHCHCOOH OH
Fumaric
HOOCCH]CHCOOH
Latic
HOOCCH=CHCOOH Acetic
CH3COOH
acids contain several carboxylic acid groups, each with a different pKa value (see Table 2). Citric acid, for example, contains three carboxylic acid groups and forms three anions, predominantly singly charged above pH 2.9, doubly charged above pH 4.3, and triply charged at above pH 5.7. This property extends the buffering capacity of citric acid, to a buffering range from pH 1.9 to pH 6.7 (Figure 3). Dicarboxylic acids also give a wide buffering range, effectively forming excellent buffers over the pH range of most acidic foods. In comparison, monocarboxylic acids possess a limited buffering range; a food acidified with acetic acid is unbuffered effectively below pH 3.75, allowing easy movement of pH in this area.
Acidification and Associated Flavor in Foods The primary chemical effect of an acidulant in food is to lower the pH value. To what extent the pH falls depends on the
buffering capacity, fat content, acid type, and concentration. The acidification ability of different acids can be compared on a molar basis or by weight. In general, food additions are determined as percentages by weight or parts per million (ppm). On a molar basis, food acidulants are surprisingly similar in acidification power (Figure 4). On a weight basis, however, differences between acids become more marked, smaller acids with lower molecular weights being most effective. Surprisingly, the flavor characteristics of these acids do not always reflect acidification power. This is because sensory cells on the tongue that detect sourness more efficiently sense acidity as a function of acid concentration (at a constant pH value), rather than the pH itself. Figure 5 shows the comparison between various acids in terms of perceived acidity, such that fumaric > malic ¼ tartaric ¼ acetic > citric > lactic.
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Citric acid Malic acid Tartaric acid Fumaric acid Lactic acid Ascorbic acid Acetic acid 1
2
3
4 5 pH value
6
7
8
Figure 3 Effective buffering ranges of acidulants commonly used in foods. Acids containing multiple carboxyl groups have broader buffering ranges than monocarboxylic acids.
Effect of Organic Acids on Microbial Cells A wide variety of acids occur naturally or are added to foods. These acids differ in structure and chemical properties (see Table 2) and different antimicrobial actions have been proposed for various acids. Figure 6 shows the likely mechanism of action by traditional acid preservatives. These include action by low pH on the cell wall and plasma membrane, action in lowering the internal cytoplasmic pH, chelation of
trace metal ions from substrate media and from the cell wall, and perturbation of membrane function by acid molecules. Some acids are antimicrobial by a single mechanism, whereas others may combine several distinct actions.
Organic Acid Acidification of Substrate Media The primary function of an acidulant is to lower the pH of foods; consequently, the primary action of traditional acid
(a)
Final pH
4.5
Equimolar acids
4.0 3.5
Ascorbic
Acetic
Phosphoric
Ascorbic
Acetic
Phosphoric
Lactic
Adipic
Fumaric
Tartaric
Malic
2.5
Citric
3.0
Acids at 25 mmol l –1
Final pH
(b)
4.5
Equal acid by weight
4.0 3.5
Lactic
Adipic
Fumaric
Tartaric
Malic
2.5
Citric
3.0
Acids at 5000 ppm., 0.5%
Figure 4 Acidification power of acidulants on an (a) equimolar or (b) weight basis. Acids were applied at 25 mmol l1 or 5000 ppm to a protein solution, 1% bacteriological peptone, initial pH ¼ 6.2.
PRESERVATIVES j Traditional Preservatives – Organic Acids
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Equivalent taste (%)
130 120 110 100 90 80 70 60 Citric
Malic
Tartaric
Fumaric
Lactic
Acetic
Acidulant acid Figure 5 Taste equivalents of acids in water, compared with citric acid (100%). Fumaric acid, being stronger tasting, requires less addition for equivalent taste.
Membrane fluidity and structure
Microbial cell External pH Cytoplasmic pH
Metal ion chelation Chelation of ions in the wall
Figure 6 Potential sites of antimicrobial action by acids. Acids may act by lowering external pH, by affecting membrane structure and fluidity, depressing cytoplasmic pH, or chelating metal ions from the substrate media or cell wall.
conformation and folding. In this regard, it has been shown that replacement of the Hþ-adenosine triphosphate (ATP)ase proton pump in yeast membranes by an ATPase pump of plant origin prevented yeast growth in acidic conditions demonstrating that the yeast pump could tolerate acidity but that the plant ATPase could not. If acidulants inhibit microorganisms only via depression of media pH, at any given pH value, all acids theoretically would be equally effective preservatives. Figure 7 illustrates that this is not true. Acetic and citric acids inhibit microbes more effectively at substantially lower concentrations than other acids, indicating that these acids possess additional mechanisms of action.
Modulation of Cytoplasmic pH by Organic Acids
Acidulant
preservatives on microorganisms involves the direct action of protons on microbial cells. Protons are charged particles that pass slowly through lipid membranes. The action of pH on microbial cells is likely to involve cell walls, the outer faces of membranes, as well as proteins protruding through the membrane. Of these, pH is the most likely to affect proteins, such as enzymes, transport permeases, and pumps. Proton association with proteins affects charge stability, altering
Lipid membranes are amphipathic and, therefore, are generally impermeable to charged ions, except by specific transport mechanisms. Protons penetrate membranes poorly, as do charged anions, and thus strong acids are often less effective antimicrobial agents in comparison with weak undissociated acids. Correspondingly, uncharged acid molecules diffuse rapidly though the plasma membrane if they are lipid-soluble. The ‘weak acid’ theory of microbial inhibition by lipophilic
Acetic acid Fumaric acid Citric acid Malic acid Tartaric acid Lactic acid Succinic acid 0
200
400
600
800
1000
Inhibitory concentration (mmol l –1) Figure 7 Comparison of the minimum inhibitory concentrations (MICs) of acidulants, determined against Saccharomyces cerevisiae X180–1B in yeast extract-peptone-dextrose growth medium (YEPD), 30 C, at pH 4.0.
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Anion
Acid
Acid Anion + Proton
pH 4.75
pH 6.5 Microbial cell
Figure 8 The ‘weak acid’ theory of microbial inhibition by acids. In media at pH 4.75, acid molecules and anions are in equimolar equilibrium. Lipophilic acid molecules diffuse rapidly through the membrane into microbial cells. The neutral cytoplasmic pH causes acids to dissociate into anions and protons, which being lipid insoluble accumulate in the cytoplasm. Excess proton accumulation eventually lowers the cytoplasmic pH.
preservatives proposes that acid molecules in foods rapidly diffuse through the plasma membrane into the cytoplasm (Figure 8). The neutral cytoplasmic pH causes acids to dissociate, by shedding Hþ into charged anions and protons, both of which are unable to diffuse out of the cell. Diffusion continues until the acid concentration is equal on both sides of the membrane, during which time, anions and protons have been concentrated in the cytoplasm. If preservatives are present at sufficient concentration, accumulated protons can overwhelm cytoplasmic buffering and lower the internal pH. Low cytoplasmic pH (pHi) then leads to denaturation of nucleic acids and enzymes, inhibits metabolism, and prevents active transport requiring a DHþ gradient. Active pumping aimed at removing protons from the cell interior by means of membrane-bound Hþ-ATPases can raise the internal pH but also can consume excessive ATP and may cause inhibition by means of energy depletion. The weak acid theory often is assumed wrongly to apply to all acids. For an acid to function as a weak acid preservative, it must satisfy the following criteria: l
Liphophilic Able to traverse (diffuse) rapidly across the cellular membrane l Concentrates within the cytoplasm as a result of low pKa l Able to release sufficient protons in the cytoplasm at the minimum inhibitory concentration (MIC) to overcome cytoplasmic buffering and depress cytoplasmic pH l
According to the partition coefficient in Table 2, traditional acidic preservatives, such as citric, malic, fumaric, and tartaric acids are not lipophilic, but rather they are very lipophobic. Consequently, these impermeant acids cannot, therefore, very well act as weak acid preservatives or depress the cytoplasmic pH, although known antimicrobial activity may be a function of external pH depression, chelation effect, and external membrane perturbation. Acetic acid, in contrast, is lipid soluble; diffuses rapidly through the plasma membrane; is efficiently accumulated in the cytoplasm; and, moreover, has been demonstrated to cause a rapid collapse in pHi. A lowering of the pH of the medium greatly enhances the effectiveness of acetic acid (Figure 9), not only increasing the undissociated acid concentration but
also increasing the degree to which anions and protons are accumulated in the microbial cytoplasm. Acetic acid appears, therefore, to exhibit antimicrobial capacity as a classic weak acid preservative, in addition to action on external pH. Lactic acid shows a degree of lipid solubility and has been shown to diffuse slowly through membranes. Inhibition by lactic acid, however, has not been correlated with a decline in pHi, and although weak acid action may contribute to inhibition by lactic acid, it appears that other mechanisms of inhibition, such as external pH depression, are also involved.
Chelation by Organic Acids Most acids form complexes with metallic ions, but for the majority, affinity of acids for metals is low, and complexes are correspondingly unstable. Certain acids, however, often those with multiple carboxylic acid groups, form stable complexes, which can chelate a substantial proportion of metallic ions, with greatest affinity for transition metal ions – for example, Fe3þ. Table 3 shows the affinity constants, K1, for acid–metal complexes. Figures quoted are the log of the equilibrium constant. Stability of citric acid complexes is some 2–3 logs greater than those of malic, tartaric, or lactic acid. The antimicrobial action by citric acid is known to involve chelation and is overcome by the addition of metal ions (e.g., Mgþþ, Caþþ). It appears probable that citric acid removes key nutrients from media, preventing microbial growth. Chelators have their greatest affinity for Fe3þ, but the identity of growth-limiting nutrients depends on microbial ion requirements, the concentrations of metal ions in media, and the affinity of acids for each ion. Action by citric acid via chelation is supported by the finding that its inhibitory action is pH dependent, with the greatest microbial inhibition occurring at higher pH values (see Figure 9). Stability of complexes formed by multiple charged anions, predominating at high pH, is some 6 logs greater than those of the undissociated acids (Table 4). The inhibitory action by malic, tartaric, and succinic acids may also involve chelation activities, given the affinity of these acids for metal ions and the high concentrations of these acids used in food. This hypothesis is corroborated in yeasts and molds, where the MIC of these acids appears to reflect the overall stability of acid–ion complexes. In addition to chelation of nutrient ions, acids also act by damaging the cell walls of bacteria by chelation of metal ions embedded within the structure. EDTA, a common chelating agent, is known to cause Gram-negative bacteria to be susceptible to a variety of antibiotics. It is thought that EDTA achieves this by removing metallic cations from the bacterial outer membrane, subsequently opening up the structure and allowing access of antibiotics to the cell.
Effects of Organic Acids on Microbial Populations and Spoilage The effect of a preservative on a microbial population may be to cause cell death, stasis (viable but inhibited cells) or retard growth. Since traditional acid preservatives include many different acids, acting by different mechanisms against a variety
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(a)
5000 4000 3000 2000
0 80 160
1000 0 2.5
3.5
4.5
5.5
6.5
7.5
Ac e (m tic a mo cid l l –1 )
Growth (mg l –1)
6000
pH value
(b)
6000 5000 4000 3000 0 200 400
2000 1000 0 2.5
3.5
4.5
5.5
6.5
7.5
Ci tr (m ic a mo cid l l –1 )
Growth (mg l –1)
7000
pH Figure 9 The effect of pH on inhibition of Saccharomyces cerevisiae X2180–1B by (a) acetic acid and (b) citric acid. Growth was examined in a matrix of flasks at pH 2.5–7.5 containing various acid concentrations, after 3 days shaken at 30 C. Acetic acid was more inhibitory at a low pH, whereas citric acid was more inhibitory at a high pH.
of microorganisms, it is understandable that there is no single, unified effect on microbial populations. Acidity – low pH – (
to acid. It is wrong, however, to assume that instantaneous bacterial death occurs in foods at a pH lower than 4.0. As incidents of food poisoning associated with Escherichia coli O157:H7 and Salmonella have shown, bacteria can remain viable in juices at pH 4.0 for several weeks and in fact actually may turn on acid resistance genes in the pathogens, whereby
Table 3 Chelation properties of acidulants used in foods; ethylenediaminetetraacetic acid (EDTA), permitted in low concentration in the United States, is listed for comparison Acid Cation
EDTA
Citric
Malic
Tartaric
Succinic
Lactic
Kþ Naþ Mgþþ Caþþ Mnþþ Znþþ Cuþþ Fe3þ
0.96 1.79–2.61 8.69 10.45–10.59 12.88–13.64 15.94–17.50 18.80–19.13 23.75–25.15
0.59 0.70 3.16–3.96 3.40–3.55 2.84–3.72 4.98 5.90 11.40
0.18–0.23 0.28–0.3 1.70 1.96 – 2.93 3.43–3.97 7.1
– 1.98 1.91 2.17 1.44–2.92 2.69–3.31 2.6–3.1 6.49
– 0.3 – 1.20 – 1.76–3.22 2.93 6.88
– – 0.73 0.90 0.92 1.61 2.5 6.4
The stability constant values, K1 for acid–metal ion complexes quoted are the log of the equilibrium constant at 20–25 C.
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Table 4 Stability constants, log equilibrium constant of copper(II) complexes with malic acid and malate ions Acid
Ligand form
Cation
Stability constant
Malic acid Malate Malate2
H2L HL L2
Cuþþ Cuþþ Cuþþ
2.00 3.42 8.00
Viable population (log cfu ml –1)
Undissociated malic acid predominates below pH 3.2, malate predominates between pH 3.2 and 4.7 and malate2 above pH 4.7
10 8 6 4 2 0
50
100
150
200
Time (min) Figure 10 Growth-phase-dependent death of Listeria monocytogenes ScottA, in BHI media acidified to pH 3.0 with HCl. Open squares, stationary phase; solid squares, exponential phase. Courtesy of MJ Davis.
they are more resistant to stomach acid during passage through the human gastrointestinal tract. Lower pH values (e.g., 2.5), furthermore, although inactivating most enteric pathogenic bacteria, have little or no effect on the viability of spoilage yeasts or molds. Weak acids that are able to diffuse through the plasma membrane, including lactic, acetic, and benzoic acids, greatly exacerbate the effect of pH. Bacterial populations are killed faster and at higher pH values when acidified with weak lipophilic acids. Growth of yeasts and molds is inhibited by permanent weak acids, usually without losing viability. Weak acids characteristically prolong lag phase duration, and at subinhibitory concentrations, reduce growth and metabolic function. Chelating agents also characteristically do not kill microorganisms but rather prevent growth by limiting metallic nutrient availability.
Sublethal Effects, Species–Strain Variability, and Interaction with Other Factors Acid Resistance and Sensitivity There is considerable variation in microorganisms as to their sensitivity to traditional acid preservatives. This variation extends from the overall sensitivity to low pH of bacteria, yeasts, and molds – to particular acid-resistant genera – to variation in sensitivity of strains within species and even variation between individual cells in populations, caused by their phase of growth and previous history. Acid-tolerant bacteria include acetic acid bacteria, such as the genera Acetobacter and Gluconobacter spp. and lactic acid
bacteria, such as Lactobacillus, Lactococcus, Streptococcus, Leuconostoc, Enterococcus, and Oenococcus. Other acid-tolerant sporeforming bacteria include the Gram-positives – Clostridium butyricum and Clostridium pasteurianum, Alicyclobacillus spp., Bacillus coagulans, Bacillus macerans, and Bacillus polymyxa. Bacterial spoilage at low pH most frequently is associated with Gram-negative bacteria belonging to the genus Gluconobacter (Acetomonas). These bacteria require oxygen for growth and are restricted by gas-impermeable packaging and minimal head space. Acetic acid bacteria are resistant to normal concentrations of preservatives. Lactobacilli and Leuconostoc spp., the lactic acid bacteria, are known to promote spoilage through loss of astringency, production of slime and gas, ropiness, turbidity, or production of off-flavors. These microorganisms can grow in products at pH 2.8 but are relatively heat sensitive. Spore-forming Alicyclobacillus (formerly Bacillus) spp. acidoterrestris, acidocaldarius, and cycloheptanicus can survive pasteurization and grow well at low pH. Consequently, these organisms are particularly problematic for the beverage industry. Even at low levels of growth, Alycyclobacillus generates the highly pungent phenolic metabolic by-product guiacol, which can be detected by the human nose in the range of parts per billion and leads to the spoilage of fruit juices, fruit juice blends, sports drinks, and lemonade. There are a number of preservative-resistant spoilage yeasts. Zygosaccharomyces bailii, Zygosaccharomyces bisporus, and Zygosaccharomyces lentus are highly preservative resistant. Zygosaccharomyces rouxii, Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces exiguus, Schizosaccharomyces pombe, and Torulaspora delbrueckii are moderately to highly resistant. Individual strains show considerable variation in acid resistance (Figure 11), despite genetic confirmation that these individual strains belong to the same species. Molds generally are more sensitive to weak acid preservatives, an exception being Moniliella acetobutens, an acetic acid–resistant mold that causes spoilage in pickles and vinegar.
Habituation and Adaptation to Acids The addition of organic acids to foods progressively lowers the pH value and increases the concentration of acid. Studies involving bacteria tend to focus on the effect of pH, this being the major bactericidal force, whereas yeasts and molds, which are substantially immune to low pH, have been studied more extensively in relation to the acid concentration. Acid stress responses, sublethal adaptation, and habituation to pH and acids are not yet fully understood and thus remain an area of active research in bacteria and yeasts. It is well established for many bacteria, including E. coli, Salmonella typhimurium, Listeria monocytogenes, and Lactobacillus spp., that survival at low pH is enhanced by prior exposure to mildly acidic pH. Bacteria cultured and transferred from neutrality to pH 3 die rapidly. An intermediate stage at pH 4.5 for some 20–60 min greatly increases the proportion of surviving cells. This acid tolerance response (ATR) is not yet fully characterized, although there appear to be several components of the ATR, some requiring protein synthesis and others which are shared in stationary phase resistance. The ATR involves a set of some 50 gene products, acting to improve pH
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800 600 400 0.2
Ac et 0.6 i (% c ac 1.0 ) id
200
Growth (mg l –1)
1000
0 D2627
1601
2406
5207
5316
Zygosaccharomyces lentus Figure 11 Variation in sensitivity to acetic acid by individual strains of Zygosaccharomyces lentus. Yeasts were grown in yeast extract-peptone-dextrose (YEPD) growth medium, corrected to pH 4.0 after acetic acid addition, at 25 C for 1 week. Courtesy of H. Steels.
homeostasis, reducing energy dissipation, enhancing DNA repair, correcting protein misfolding by chaperones, and continuing membrane biosynthesis. The rpoS gene encodes an alternative sigma factor ss, a critical stationary phase regulator that is also involved in the ATR. The protein RpoSp is synthesized semiconstitutively and typically degrades rapidly. When the growth rate is impaired, the regulatory ss is stabilized and induces expression of a number of enzymes involved in protection and repair of DNA and proteins and in detoxification. Adaptation by yeasts to acid preservatives has been an ongoing industrial problem for decades. Growth of yeasts on splashes of preserved products results in populations of adapted yeasts capable of tolerating unusually high concentrations of preservatives. Figure 12 shows that yeasts grown for 1 week at subinhibitory concentrations of acetic acid subsequently can grow in media containing twice the concentration of acetic acid. The explanation for adaptation by yeasts may involve
Interaction of Acidulants with Other Factors The most significant factors capable of modifying the preservative effect of acidulants are the pH and the intrinsic buffering capacity of the food. The primary antimicrobial action by acidulants is to lower the pH. Foods with a higher pH or with substantial buffering will limit pH reductions. Buffering may be achieved by other acids or their corresponding salts or by the presence of substantial quantities of proteins or amino acids. Lowering the pH substantially increases the effect of lipophilic acetic acid, acting as a weak acid preservative but may decrease the effect of chelating acids such as citric acid (see Figure 9). (b)
1600
1600
1400
1400
1200
1200 Growth (mg l –1)
Growth (mg l –1)
(a)
mechanisms to conserve ATP, pdr12 drug-resistance pumps to remove acid, or simply that accumulation of acids within the cytoplasm creates buffering capacity (see Figure 8), which resists further change in pHi when cells are reinoculated into higher levels of acetic acid.
1000 800 600
1000 800 600
400
400
200
200
0
0 0
30
60
90
120
Acetic acid (mmol l –1)
150
180
0
30
60
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150
180
Acetic acid (mmol l –1)
Figure 12 Adaptation by Saccharomyces cerevisiae X2180-1B to acetic acid in yeast extract-peptone-dextrose (YEPD) growth medium, at pH 4.0, 30 C. (a) Nonadapted yeasts were grown for 7 days in tubes of YEPD containing acetic acid. Adapted yeasts were taken from the highest concentration of acetic acid permitting growth (90 mmol1) and reinoculated into a similar series of tubes. (b) Growth of adapted yeast was measured after further 7 days.
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Many foods are preserved by high-temperature pasteurization. When acidulants are used to lower the pH, the pasteurization requirement is reduced substantially. This partially is due to the effect of acids on bacterial spores. Heat-resistant bacterial spores often require heating to 121 C for many minutes to achieve sterilization. At pH values below 4.0, spore germination is inhibited and pasteurization at a much lower temperature is sufficient to kill vegetative bacterial and yeast cells. Additionally, heat is a much more effective sterilant under acidic conditions, a property that is capitalized upon during traditional and commercial fruit juice bottling. The behavior of acids in foods also depends on the nature and properties of the food itself. Foods contain proteins, composed of amino acids, which effectively buffer the food matrix, resisting changes in pH. Foods also may contain fats or lipids. Lipophilic acids may be removed from solutions by partitioning them into the lipid fractions. The partition coefficients for food acidulants are shown in Table 2, as log Poct values, the log of the distribution between water and octanol. Negative values show that acids are preferentially soluble in water, rather than lipids. Lactic acid and acetic acid, however, are moderately lipophilic and a sizable fraction of these acids may partition into the lipid phase in foods containing fats or oils (e.g., salad cream, mayonnaise, and dressings) and can be reduced effectively in concentration.
See also: Ecology of Bacteria and Fungi in Foods: Influence of Redox Potential; Ecology of Bacteria and Fungi in Foods: Effects of pH; Spoilage Problems: Problems Caused by Bacteria; Spoilage Problems: Problems Caused by Fungi; Zygosaccharomyces; Preservatives: Permitted Preservatives – Benzoic Acid; Preservatives: Permitted Preservatives – Sorbic Acid.
Further Reading Baird-Parker, A.C., Kooiman, W.J., 1980. Soft Drinks, Fruit Juices, Concentrates, and Fruit Preserves, Microbial Ecology of Foods. Food Commodities, vol. 2. Academic Press, London, p. 643. International Commission on Microbiological Specifications for Foods. Bearso, S., Bearson, B., Foster, J.W., 1997. Acid stress responses in enterobacteria. FEMS Microbiology Letters 147, 173–180.
Beuchat, L.R., Golden, D.A., 1989. Antimicrobials occurring naturally in foods. Food Technology 43, 134–142. Booth, I.R., Stratford, M., 2000. Acidulants and low pH. In: Russell, N.J., Gould, G.W. (Eds.), Food Preservatives, second ed. Blackie, Glasgow. Chichester, D.F., Tanner, F.W., 1972. Antimicrobial food additives. In: Furia, T.E. (Ed.), Handbook of Food Additives, second ed. CRC Press, Cleveland, p. 155. Corlett, D.A., Brown, M.H., 1980. pH and Acidity, Microbial Ecology of Foods. Factors Affecting Life and Death of Microorganisms, vol. 1. Academic Press, London. p. 92. International Commission on Microbiological Specifications for Foods. Doores, S., 2005. Organic acids. In: Davidson, P.M., Sofos, J.N., Brannen, J.L. (Eds.), Antimicrobials in Foods, third ed. CRC Press, Taylor & Francis Group, Boca Raton, FL, pp. 91–142. EEC, 1989. Council Directive on Food Additives Other than Colours and Sweeteners. EC, Brussels (89/107/EEC). Eklund, T., 1989. Organic acids and esters. In: Gould, G.W. (Ed.), Mechanisms of Action of Food Preservation Procedures. Elsevier Applied Science, London, p. 161. Gardner, W.H., 1972. Acidulants in food processing. In: Furia, T.E. (Ed.), Handbook of Food Additives, second ed. CRC Press, Cleveland, p. 225. Ingram, M., Ottaway, F.J.H., Coppock, J.B.M., 1956. The preservative action of acidic substances in food. Chemistry and Industry (London) 75, 1154–1163. Kabara, J.J., Eklund, T., 1991. Organic acids and esters. In: Russell, N.J., Gould, G.W. (Eds.), Food Preservatives. Blackie, Glasgow, p. 44. Pitt, J.I., Hocking, A.D., 1997. Fungi and Food Spoilage, second ed. Blackie Academic and Professional, London, p. 439. Somogyi, L.P., 1996. Direct food additives in fruit processing. In: Somogyi, L.P., Ramaswamy, H.S., Hui, Y.H. (Eds.), Processing Fruits: Science and Technology. Biology, Principles and Applications, vol. 1. Technomic, Lancaster, p. 293. Stratford, M., Eklund, T., 2000. Organic acids and esters. In: Russell, N.J., Gould, G.W. (Eds.), Food Preservatives, second ed. Blackie, Glasgow. Taylor, R.B., 1998. Ingredients. In: Ashurst, P.R. (Ed.), The Chemistry and Technology of Soft Drinks and Fruit Juices. Sheffield Academic Press, Sheffield, p. 16. Theron, M.M., Ryekers Lues, J.F. (Eds.), 1996. Organic Acids and Food Preservation. CRC Press, Taylor & Francis Group, Boca Raton, FL, p. 318. Zhao, T., Zhao, P., Doyle, M.P., 2009. Inactivation of Salmonella and Escherichia coli O157:H7 on lettuce and poultry skin by combinations of levulinic acid and sodium dodecyl sulfate. Journal of Food Protection 72, 928–936.