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PRESERVATIVES | Classification and Properties
PRESERVATIVES
Contents Classification and Properties Permitted Preservatives – Benzoic Acid Permitted Preservatives – Hydroxybenzoic Acid Permitted Preservatives – Natamycin Permitted Preservatives – Nitrites and Nitrates Permitted Preservatives – Propionic Acid Permitted Preservatives – Sorbic Acid Permitted Preservatives – Sulfur Dioxide Traditional Preservatives – Oils and Spices Traditional Preservatives – Organic Acids Traditional Preservatives – Sodium Chloride Traditional Preservatives – Vegetable Oils Traditional Preservatives – Wood Smoke
Classification and Properties M Surekha and SM Reddy, Kakatiya University, Warangal, India Ó 2014 Elsevier Ltd. All rights reserved.
Introduction Fresh foods always contain microorganisms both on their surfaces and within. These microorganisms, if they are not destroyed, will spoil the food. The prevention of food spoilage by inhibiting or destroying the microorganisms is the basis of food preservation. This can be done by chemical treatment, freezing, curing, dehydration, or thermal processing. The chemicals used to prevent food spoilage have some antiseptic properties under the conditions of use and are known as preservatives. Broadly speaking, a preservative is a chemical substance capable of retarding or arresting the growth of microorganisms to prevent such processes as fermentation, acidification, or decomposition, which cause deterioration of flavor, color, texture, appearance, and nutritive value. The main objectives of using preservatives are to extend the shelf life, retain nutritive value, and ensure safety. Chemical preservatives often are used in combination with physical methods; such combinations may allow the preservatives to be used at lower concentrations, thus retaining the quality of the product.
The Need for Preservatives The twentieth century witnessed radical technological advancement in the physical methods of food preservation.
Encyclopedia of Food Microbiology, Volume 3
These developments include preservation of food by thermal processing, refrigeration, freezing, concentration, drying, and more recently the use of irradiation. In spite of this technological advancement, the worldwide population explosion has resulted in a crisis of food supply, which demands a reduction in losses to the minimum. The countries with the greatest nutritional need are the least developed, suffering from inadequate production, distribution, transportation, storage, and preservation facilities. These countries are not in a position to afford the latest technologies for the preservation of food by physical methods, and thus they depend on the use of chemical preservatives that are not only effective but also safe and inexpensive. As physical methods are not suitable for all types of foods, these days even industrial countries are making use of chemical preservatives.
Properties of Preservatives The desirable properties of a chemical substance to serve as a preservative are as follows: 1. A preservative used for antimicrobial purposes should kill the microorganisms rather than inhibit their growth. 2. Any bacteriostatic preservative is most effective if it persists until the food is ready for consumption. If the food is undergoing processing, the bacteriostatic preservative should persist until the food is further processed.
http://dx.doi.org/10.1016/B978-0-12-384730-0.00257-3
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PRESERVATIVES j Classification and Properties
3. A preservative should have an adequate degree of resistance to heat. 4. The specificity range of a preservative should correspond with the range of microorganisms that contaminate and develop on the food. 5. A preservative that is intended to supplant thermal processing should provide a degree of security against Clostridium botulinum similar to that given by the normal thermal processing. 6. The preservative should neither be destroyed by the miscellaneous reactions of the food nor be inactivated by the metabolic products produced by the microorganism. 7. Any antimicrobial preservative should not readily stimulate the appearance of resistant strains of microorganisms. 8. There should be a suitable procedure for determining the amount of the preservative in different foods.
Table 1
Preservatives used in food
Traditional preservatives Sugar Salt Smoke Spices Vinegar Alcohol
Other desirable properties of a preservative are as follows: l
It should have a practical value and be economical. It should be a nonirritant and have low (or no) toxicity. l It should not retard the activity of digestive enzymes or harm the consumer. l Within the body, it should not decompose into substances more toxic than the preservative itself. l
Synthetic preservatives
Bacteriocins
Organic Acetic acid, acetates, and diacetates Sorbic acid and its salts Benzoic acid and its salts p-hydroxybenzoic acid esters and their salts Boric acid and borates Citric acid and its salts Formic acid and formates Lactic acid and its salts Propionic acid and its salts Inorganic Polyamino acids Carbonic acid (CO2) Sulfurous acid and sulfites (SO2) Nitrites and nitrates Phosphates Hydrogen peroxide
Nisin
Bacteriocins
Classification Preservatives include traditional (natural) preservatives, bacteriocins, and synthetic preservatives.
Traditional Preservatives Compounds such as sugar, salt, vinegar, organic fruit acids, wood smoke, alcohol, and various spices used in the preservation of food for centuries are regarded as traditional preservatives. Salts and sugars dissolve in the water of the food to form strong solutions in the process of curing and conserving. The difference between the concentration of the solution and that of the microbial cell cytoplasm causes dehydration of the cell, which leads to its inhibition or death. Salamis, hams, jams, and condensed and sweetened milk are examples of this principle. Smoking destroys bacteria on the surface of food.
Synthetic Preservatives Apart from vinegar, some other acids and their salts are legally permitted preservatives (Table 1). The other synthetic preservatives used are nitrites and nitrates, sulfur dioxide, and sulfites, carbon dioxide, phosphates, and hydrogen peroxide. Chemical preservatives are classified based on their chemical nature and action. On the basis of their chemical nature, they are of two types: inorganic preservatives and organic preservatives. Nitrates, nitrites, sulfites, sulfurous acid, borates, hypochlorites, and peroxide are inorganic preservatives. Benzoates, formic acid, sorbic acid, and propionic acid – and their sodium and calcium salts – as well as esters of p-hydroxybenzoic acid are classified as organic preservatives.
Bacteriocins are a group of small antimicrobial peptides and mostly are plasmid mediated. They generally inhibit only closely related bacteria. Species and strains of Gram-positive lactic acid bacteria (LAB) possess the capacity to produce bacteriocins or bacteriocin-like compounds. Bacteriocins have attracted particular attention as their producer organisms have GRAS (generally recognized as safe) status and are naturally present in many food products. Bacteriocins are a heterogenic group of peptides and can be grouped into the following three classes: 1. Lantibiotics (with 19–37 amino acids), heat stable-Nisin, Lactocin S, Lacticin 3147, and Subtilin 2. Nonlantibiotics (<15 kDa), small and heat stable-Pediocin PA-1, Lactacin B, Lactacin F, Leucocin A-UAL 187, and Lactococcin G 3. Small, heat labile proteins of more than 30 kDa – Caseicin 80, Lacticins A and B Out of many bacteriocins, Nisin is the only purified bacteriocin extensively used as food preservative in many countries. Nisin is a polypeptide with molecular weight 3500 Da, with its rare amino acids (Lanthionine, 3-methyl-lanthionine, dehydroalanine, and dehydrobutyrine). Nisin has several advantages as a food preservative as it is nontoxic, easily degraded by digestive enzymes, thermostable, and does not contribute offflavors and off-odors. Nisin initially forms a complex with a lipid precursor molecule in the formation of bacterial cell walls. The Nisinlipid complex-II then inserts itself into cytoplasmic effuse of essential cellular components, resulting in inhibition or death of bacteria. Gram-negative bacteria are resistant to Nisin because their outer membrane, which is making cell walls, is far less permeable than those of Gram-positive bacteria.
PRESERVATIVES j Classification and Properties Table 2
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Inhibitory action of sorbic acid, benzoic acid, and sulfur dioxide on bacteria, yeasts, and molds Preservatives Sorbic acid
Organism Bacteria Escherichia coli Serratia marcescens Bacillus sp. Clostridium sp. Salmonella sp. Lactobacillus sp. Pseudomonas sp. Streptococcus sp. Micrococcus sp. Yeasts Saccharomyces sp. Hansenula anomala Torulopsis sp. Candida krusei Candida lipolytica Byssochlamys fulva Molds Rhizopus Geotrichum candidum Oospora lactis Penicillium sp. Aspergillus sp. Fusarium sp. a
Benzoic acid a
SO2 a
pH
MICa
pH
MIC
pH
MIC
5.2–5.6 6.4 5.5–6.3 6.7–6.8 5.0–5.3 4.3–6.0
50–100 50 50–1000 100–1000 50–1000 200–700
5.2–5.6
50–120
100–200 50
4.3–6.0 6.0 5.5–5.6 5.2–5.6
300–1800 200–400 50–100 200–400
100
3.2–5.7 5.0 4.6 3.4 5.0 3.5
30–100 500 400 100 100 50–250
3.6 4.8 3.5–4.5 3.5–5.7 3.3–5.7 3.0
120
5.0
25–200 20–100 20–100 100
2.6–5.0 3.0–5.0
200–300 200–500 300–700
30–120 1000 300 30–280 20–300
4.0 5.0
80–160 240
5.0 4.5
160–400 220
MIC, minimum inhibitory concentration, expressed in parts per million (ppm).
Antimicrobial Properties Spectrum of Activity These preservatives do not have a complete spectrum of action against all microorganisms that spoil foods. Most preservatives predominantly act against yeasts and molds (Table 2). In general, most of the organic acids have the broadest spectrum of antimicrobial activity and are useful against many spoilage bacteria, fungi, and yeasts. Benzoic acid is used primarily as an antimycotic agent and most yeasts and molds are inhibited. The activity of benzoic acid against bacteria is variable. Propionic acid and its salts are highly effective mold inhibitors, but yeasts and most bacteria are less affected. Inhibition of ropeforming bacteria in bread is a specific target for propionic acid. Acetic acid is more effective against yeasts and bacteria than molds; Acetobacter sp., certain LAB, and some yeasts are resistant to acetic acid. Lactic and citric acids have only moderate antimicrobial activity. These acids inhibit the formation of aflatoxin and sterigmatocystin. Sorbic acid and its salts have a wide spectrum of activity against catalase-positive bacteria, yeasts, and molds and are highly active against osmophilic yeasts. Sulfur dioxide and sulfites also have a broad spectrum of antimicrobial activity in acid foods. This preservative is more effective against bacteria than molds and yeasts, with Grampositive bacteria being less susceptible than Gram-negative bacteria. Sulfites inhibit enterobacteria and Salmonella. Lactobacilli are highly sensitive to SO2. Yeasts react differently to SO2 depending on the strain. The practical importance of nitrite is in the inhibition of spore-forming bacteria; it also affects
Achromobacter, Aerobacter, Escherichia, Flavobacterium, Micrococcus, and Pseudomonas.
Mechanism of Antimicrobial Action Food preservatives inhibit not only the general metabolism but also the growth of the microorganisms. Depending on the type of preservative used, the final state at which the microorganisms are killed is reached within a few days or weeks, at the usual applied concentrations. The timescale for the killing of microorganisms under the influence of preservatives corresponds to the relationship K ¼ 1=t$ln Z0 =Zt or Zt ¼ Z0 $eKt where K is the death rate constant, t1 is the time period, Z0 is the number of living cells at the time when the preservative begins to act, and Zt is the number of living cells after time t. The given formula is considered to be the basis for studying the action of preservatives in foods. This rule is valid, however, only for relatively high dosages of preservatives and a genetically uniform cell material. A preservative added to a food when microbial counts are low inhibits microorganisms in the initial lag phase; the dosage of preservatives necessary in practice to inhibit microorganisms in the exponential log phase would be too high. Preservatives are not designed to kill microorganisms in substrates already supporting a massive
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PRESERVATIVES j Classification and Properties
germ population. In general, the action of preservatives includes physical as well as physicochemical mechanisms, especially the inhibitory action on enzymes. The partial dissociation of weakly lipophilic acid food preservatives plays an important role in the inhibition of microbial growth. The undissociated lipophilic acid molecules are capable of moving freely through the membrane. They pass from an external environment of low pH (where the equilibrium favors the undissociated molecules) to the cytoplasm, which is of high pH (where the equilibrium favors the dissociated molecules). At the high pH level, the acid ionizes to produce protons, which in turn acidify the cytoplasm and break down the pH component of the proton motive force. To maintain the internal pH, the cell then tries to expel the protons entering it. In doing so, it diverts the energy from growthrelated functions and hence both the growth rate and yield of the cell fall. If the external pH is low and the extracellular concentration of the acid is high, then the cytoplasmic pH drops to a level at which growth is no longer possible and the cell eventually dies. Some preservatives also exert specific effects on metabolic enzymes. Sorbic acid is reported to react with the sulfhydryl groups of enzymes, such as fumarase, aspartase, succinic dehydrogenase, catalase, and peroxidases in bacteria, molds, and yeasts. Antimicrobial activity of organic acids increases with chain length, but the limited water solubility of long-chain acids restricts their use. Benzoic acid is effective only in acid foods. It inhibits enzymes of acetic acid metabolism, oxidative phosphorylation, amino acid uptake, and various stages in the tricarboxylic acid cycle. It also alters membrane permeability of the microbial cell. Transport inhibition is the primary mode of action of parabens. Respiration of microbial cells also is inhibited. Antimicrobial action of propionic acid is due to inhibition of nutrient transport and growth by competing with substances like alanine and other amino acids required by microorganisms. Antimicrobial action of formic acid is similar to any acidulant. Additionally, formic acid inhibits decarboxylase and heme enzymes, especially catalase. The antimicrobial effect of other acids (e.g., lactic, tartaric, phosphoric, and succinic acids) is due to acidification of the microbial cell and inhibiting nutrient transport. Sulfur dioxide is highly reactive, and therefore it interacts with many cell components. The sulfite ion acts as a powerful nucleophile, cleaving the disulfide bonds of proteins, which changes the molecular configuration of enzymes, thus modifying active sites. It reacts with coenzymes (nicotinamide adenine dinucleotide (NADþ)), cofactors, and prosthetic groups such as flavin, thiamin, heme, folic acid, and pyridoxyl. In the case of yeast, the blocking of the oxidation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate is the salient feature. Sulfite treatment of yeast cells results in a rapid decrease in adenosine triphosphate (ATP) content prior to cell death. This is attributed to inactivation of the enzyme glyceraldehyde-3-phosphate dehydrogenase. Sulfite also reacts with carbonyl constituents of the metabolic pool to form hydroxysulfonates. Yeasts when treated with sublethal concentrations of sulfite tend to excrete increased amounts of acetaldehyde. This is due to the trapping of this metabolic intermediate as the stable hydroxysulfonate, thereby preventing its conversion to ethanol so that the reaction equilibrium shifts. Glycerol is
formed instead of ethanol by reduction of glyceraldehyde-3phosphate to glycerol-3-phosphate, which subsequently is dephosphorylated. In Escherichia coli NAD-dependent formation of oxalacetate from malate is inhibited. Sulfite destroys the activity of thiamin by breaking the bond between the pyrimidine and thiazole portion of the molecule. The antimicrobial action of nitrite is based mainly on the release of nitrous acid and oxides of nitrogen. Nitrite inhibits active transport of proline in E. coli and aldolase from E. coli, Enterococcus faecalis, and Pseudomonas aeruginosa. Reaction between nitric oxide from the nitrite and iron of a cidophore compound involved in electron transport in clostridia accounts for the anticlostridial action. Nitrite reacts with heme proteins such as cytochromes and sulfhydryl enzymes, resulting in the formation of S-nitroso products.
Combination of Preservatives No single preservative is active against all spoilage microorganisms. Attempts have been made to compensate for this by combining various preservatives with different spectra of action. In general, organic acids are compatible with other preservatives and many combinations are synergistic, for example, the following: l
Benzoate with SO2, CO2, NaCl, boric acid, or sucrose Propionate with CO2 or sorbate l Sorbate with sucrose or NaCl l Lactic acid with acetic acid. l
The combinations of sorbic acid, benzoic acid, or esters of phydroxybenzoic acid with Nisin and tylosin are useful because they extend the spectrum of action to cover E. coli, Lactobacillus, and Staphylococcus strains. Cured meats are rarely involved in Clostridium perfringens food poisoning. This is a fine example of the hurdle concept: Individual preservatives such as salt content, nitrite, and heat processing are insufficient to ensure safety but effectively control growth of C. perfringens in combination. Contrary to general expectations, not all combinations give better results than individual constituents. The presence of one preservative may sometimes weaken the effect of the other. For instance, boric acid has the tendency to weaken the effect of other preservatives in their action on E. coli. Its action against fungi proved to be synergistic, however. On the other hand, the presence of some chemical substances such as calcium chloride, which is not a food preservative and has no antimicrobial effect individually, slightly weakens the efficacy of sorbic acid, benzoic acid, and other preservatives. In general, a beneficial effect will be obtained by using preservatives with substances that counter dissociation, such as acids, or those that reduce water activity, for example, NaCl or sugar.
Degradation of Preservatives In general, food preservatives are stable substances and are unlikely to decompose within the specified storage time. Occasionally, however, certain preservatives such as organic compounds are decomposed by microorganisms and are used as a source of carbon by them. Decomposition of this type of preservative is possible, if the preservative is ineffective against microbes and also if the food contains a large number of
PRESERVATIVES j Classification and Properties microbes. Therefore, it is impossible with such preservatives to arrest the spoilage of food and to maintain the food in an apparently fresh condition. The best example of this phenomenon is the conversion of sorbic acid to hexadienol by some strains of LAB. This product reacts with ethanol to form 1ethoxy-2,4-hexadiene and 2-ethoxy-3,5-hexadiene, which give a geranium-type odor in wines.
Interaction of Preservative with Food Components Chemical reaction between food preservatives, food components, and microorganisms may lead to the formation of reaction products of toxicological importance and reduction in the concentration and the activity of the preservative. Some food preservatives such as sorbic acid, SO2, sulfites, and nitrites have an extensive reactivity with food components. Sorbic acid reacts with low-molecular-weight thiols of food, such as cysteine and glutathione, to form the 5-substituted 3hexenoic acid. Sorbic acid also undergoes autooxidation to malonaldehyde, acetaldehyde, and b-carboxyacrolein. Owing to its high chemical reactivity, sulfur dioxide may be involved in a variety of interactions with food ingredients. The action of SO2 in destroying thiamin in food is significant. An important nucleophilic reaction of the sulfite ion is its addition to the a-b-unsaturated carbonyl moiety of 3,4-dideoxyosulos3-enes formed as reactive intermediates in Maillard and ascorbic acid browning, which causes a considerable depletion of the preservatives in foods susceptible to nonenzymatic browning. The nitrite added to meat is converted to nitric oxide, which combines with myoglobin to form nitric oxide myoglobin. The N-nitrosamines formed by the cooking of nitrite-cured meat are potent carcinogens. Nitrosophenols formed by C-nitrosation of phenolic components of food are readily oxidized to the corresponding nitro compounds; S-nitroso compounds are readily formed by the nitrosation of thiols and represent a reversibly bound form of the preservative.
Uses Preservatives applicable to a particular need are determined by the composition of the food, the type of microbial spoilage, and the desired shelf life. As well as the specific physical properties, cost is also an important consideration in the selection of preservative. Preservatives are incorporated directly into food products or developed during processing food. Traditional preservatives have been introduced through processes such as fermentation, salting, curing, and smoking. Spices are commonly added in small amounts to the food as a preservative. Sugar is used in the preservation of jams, jellies, candied fruit, and sweetened condensed milk. The main use of smoke is to preserve meat and fish products. Salt is used to preserve many foods, including butter, margarine, cheese, sausages, ham, and fish. Chemical preservatives may be applied directly, most often as an ingredient of manufactured foods, but also by dipping, spraying, gassing, or dusting. Some preservatives are incorporated in the packing material rather than applied directly to the food. Vinegar or acetic acid is used in many foods, including
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mayonnaise, catsup, salad dressings, pickles, and meat. Lactic acid is used as a flavoring agent in frozen desserts and as an emulsifier in bakery products. It is also used for faster nitrite depletion and botulinal protection due to lowered pH in meat product processing. Sodium benzoate is the most widely used preservative for acid foods, including carbonated and still beverages, salads, fruit desserts, fruit cocktails, and margarine. Sodium benzoate is used at concentrations of 0.03–0.10%. Because of the astringent flavor of benzoates, they often are used in combination with sorbate or parabens. Parabens are used to preserve soft drinks, fruit products, jams, jellies, pickles, cream, and pastes. The N-heptyl ester can be used in beer fermentation at a level of 12 mg g1. Parabens may be added as a dry or liquid ingredient to food. Sorbic acid and its salts frequently are used because of their high solubility. Sorbates are used in cheese products, baked foods, fruits, fruit juices, vegetables, soft drinks, wines, jellies, jams, syrups, salads, margarine, and fish products. Since sorbate inhibits yeasts, it is not used in yeast-raised bread. Sorbate may be added directly to the food or it may be applied by dipping, spraying, dusting, or impregnating packing materials and wrappers. Recent studies showed the effectiveness of sorbate as an antibotulinal agent in meat products. In food processing, gases such as SO2 and CO2 may be used as antimicrobial agents or for other purposes. These gases may have direct or indirect antimicrobial effects. Sulfur dioxide is principally used in wine preservation. It is employed as a liquid under pressure or in aqueous solution. Additionally, various sulfite salts (sodium sulfite, sodium hydrogen sulfite, sodium metabisulfite, potassium metabisulfite, and calcium sulfite) containing 52–68% active SO2 are used to preserve a variety of foods, such as fruit juices, soft drinks, dehydrated fruits and vegetables, pickles, syrups, meat, and fish products. In wine, sulfite also is used as an equipment sanitizer, antioxidant, and clarifier and to prevent bacterial spoilage during storage. A combination of 200 mg sorbic acid, 220 mg potassium sorbate, and 20–40 mg SO2 per liter provides a comprehensive protection to the wine. Proteolytic breakdown of meat may be prevented by sulfites. Sulfur dioxide is added to foods to prevent enzymatic reactions, notably browning. Carbon dioxide is used to control psychrotrophic spoilage of meat and meat products, poultry, fish, eggs, fruits, and vegetables. Carbon dioxide generally is used in the form of a liquefied gas or as dry ice (solid CO2), which sublimes to form CO2 gas. Carbon dioxide applied under pressure with low temperature results in rapid biocidal action. Carbon dioxide is a major applicant in carbonized soft drinks, mineral water, wines, beers, and ales. It functions as an antimicrobial and effervescing agent. It inhibits aerobic spoilage organisms when used in vacuum-packed meats at a concentration of 10–20%; higher concentrations may cause undesirable odors. The combination of O2 and CO2 in a controlled atmosphere delays the respiration, ripening, and spoilage of stored fruits and vegetables. Nitrites are added to cheese and meat products. The addition of nitrite not only prevents the growth of toxigenic microorganisms but also the production of toxins. Nitrite added to meat results in both chemical and antimicrobial effects. It reacts with heme proteins to form the characteristic
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PRESERVATIVES j Classification and Properties
cured meat color and has a mild antioxidant effect that prevents rancidity and a warmed-over flavor. At low pH, nitrite is depleted by increased formation of nitrous acid and nitric oxides, which are the reactive forms of nitrite. Because of this, the addition of acids, acidulant, or glucono-delta-lactone has a beneficial effect on the action of nitrite. For the positive chemical effect (color and flavor), a nitrite concentration of 50 mg g1 is needed, whereas antibotulinal activity requires a concentration of 100 mg g1. Lower concentrations of nitrite (40–80 mg g1) in combination with sorbate are more effective. In the United States, the content of sodium nitrite in cured meat products is limited to 200 mg g1, with specific regulatory levels varying with the product. In the United Kingdom, potassium and sodium nitrite are permitted in cured meats up to a maximum of 200 mg g1. Nisin has been used to preserve dairy products, egg products, pasteurized soups, flavor-based products, canned foods, meat products, sea foods, salad dressing, and alcohol beverages. Nisin is predominantly sporostatic rather than sporicidal and, for this reason, it is widely used as a natural preservative. Ethylenediaminetetraacetic acid enhances the antimicrobial activity of Nisin against Gram-negative bacteria. Nisin is stable at pH 2.0 and can be autoclaved at 121 C. Increasing alkalinity results in the loss of antimicrobial activity of Nisin. Nisin is used in canned products as a sterilizing auxiliary. Natamycin (pimaricin) is permitted in some western European countries for surface preservation of cheese and as an additive to cheese coating. It has been used to retard yeast and mold spoilage of fruit, fruit juices, cottage cheese, poultry products, and sausage.
Toxicology and Regulatory Status Traditionally processed food in general finds ready acceptance by regulatory authorities. This is not the case for foods processed by the addition of chemical preservatives, where it is essential to ensure that the preservative used does not become a health hazard to human beings. Benzoic acid and its salts have low toxicity in experimental animals and humans. Humans have a high tolerance to sodium benzoate because of a detoxifying mechanism, in which benzoate and glycine or glycuronic acid are conjugated and excreted as hippuric acid or benzoyl glucuronide. Benzoate is not mutagenic in Drosophila or Salmonella but interacts with nucleosides and DNA in vitro. Sodium benzoate and benzoic acid are GRAS at concentrations up to 0.1% in the United States. In the United Kingdom, benzoic acid and its salts are permitted on a wide scale in accordance with the Preservatives in Food Regulations of 1979. Parabens toxicity is low, with an acute toxicity dose LD50 of 180–8000 mg kg1 of body weight in experimental animals, varying with the form of administration. The acceptable daily intake (ADI) is 10 mg kg1 of body weight of average human. The methyl and propyl parabens are GRAS in the United States, with a total addition limit of 0.1%. Propionic acid and its salts are readily absorbed by the digestive tract owing to their high water solubility. This acid decomposes in mammals by linkage with coenzyme A via methylmalonyl-CoA, succinyl-CoA, and succinate to yield CO2 and H2O. The ADI set by the Food and Agriculture
Organization (FAO) and the World Health Organization (WHO) is not limited. These preservatives are permitted for use in many countries. Sorbic acid is nontoxic and is metabolized by fatty acid oxidation, pathways common to both laboratory mammals and humans. The oral LD50 for rats is 7–10 g kg1 of body weight, and 6–7 g kg1 of body weight for the sodium salt. In highly sensitive individuals, this preservative irritates the mucous membranes. Sorbates have no mutagenic, teratogenic, or carcinogenic action. The FAO/WHO acceptable daily intake of sorbic acid and its salts is 25 mg kg1 of body weight, the highest ADI of the common preservatives. In the United States, sorbic acid and sorbates are GRAS. The maximum permissible level is 0.1–0.2%. These compounds are permitted in all countries for preservation of a wide variety of foods. Sulfur dioxide and sulfites in the body are oxidized to sulfate and excreted in urine. Although vitamin B1 deficiency, diarrhea, organ damage, and decreased usage of dietary protein and fat are some of the adverse effects of SO2 in human beings, actual poisoning by SO2 and sulfite is not possible because of vomiting. Sulfite is not a carcinogen, but SO2 is mutagenic. Levels of application are restricted to 500 mg g1 owing to flavor problems. The FAO/WHO acceptable daily intake of SO2 and sulfites is 0.7 mg kg1 of body weight per day for the average human. It is difficult to estimate an average human intake of SO2 since consumption of treated foods is high. Sulfur dioxide intake may sometimes exceed the ADI value. For example, the consumption of about three glasses of wine per day alone leads to an SO2 intake exceeding the ADI. Sulfur dioxide destroys the thiamin in foodstuffs, and many of the reported toxicity problems are symptoms of thiamin deficiency. It also interacts with folic acid, vitamin K, and certain flavins and flavoenzymes. This problem can be overcome by supplementing nutritionally adequate diet, which can withstand substantial intakes of SO2 in terms of thiamin destruction. Humans ingesting up to 200 mg SO2 per day showed no signs of thiamin deficiency or changes in urinary excretion. Products of nitrite, which reduces hemoglobin and increases the methaemoglobin content of the blood, are highly toxic to humans. Methaemoglobinemia may result in death due to oxygen shortage. Infants less than 6 months old are particularly susceptible. Neither nitrate nor nitrite have teratogenic action. The formation of potent carcinogenic compounds, nitrosamines, in cooked cured meat products can be reduced by the combination of nitrite with other preservatives, such as sorbic acid or common salt. The LD50 of nitrite for human beings is 300 mg kg1 body weight. Sodium and potassium nitrite are permitted in many countries, including the United States and the United Kingdom, to preserve meat and fish products and cheese. Nitrate contributes little or no preservative action except as a source of nitrite (e.g., following reduction by Micrococcus species in curing or fermenting meats). The FAO and WHO acceptable daily intake of nitrate is 0–5 mg kg1 per day and for nitrite 0–0.2 mg kg1 per day.
See also: Clostridium: Clostridium botulinum; Preservatives: Traditional Preservatives – Oils and Spices; Traditional
PRESERVATIVES j Classification and Properties
Preservatives: Sodium Chloride; Preservatives: Traditional Preservatives – Organic Acids; Preservatives: Traditional Preservatives – Wood Smoke; Permitted Preservatives: Sulfur Dioxide; Preservatives: Permitted Preservatives – Benzoic Acid; Permitted Preservatives – Hydroxybenzoic Acid; Permitted Preservatives: Nitrites and Nitrates; Preservatives: Permitted Preservatives – Sorbic Acid; Preservatives: Permitted Preservatives – Nisin; Permitted Preservatives – Propionic Acid; Preservatives: Traditional Preservatives – Vegetable Oils.
Further Reading Adams, M.R., Moss, M.O., 1996. Food Microbiology. New Age International, New Delhi. Branen, A.L., Davidson, P.M. (Eds.), 1983. Antimicrobials in Food. Marcel Dekker, New York.
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Branen, A.L., Davidson, P.M., Salminen, S. (Eds.), 1990. Food Additives. Marcel Dekker, New York. Busta, F.F., Foegeding, P.M., 1983. Chemical food preservatives. In: Block, S.S. (Ed.), Disinfection, Sterilization and Preservation, third ed. Lea and Febiger, Philadelphia, pp. 656–694. Davidson, P.M., Branen, A.L. (Eds.), 1993. Antimicrobials in Foods, second ed. Marcel Dekker, New York. Gould, G.W. (Ed.), 1989. Mechanism of Action of Food Preservation Procedures. Elsevier, London. Hayes, P.R., 1985. Food Microbiology and Hygiene. Elsevier, London. ICMS, 1980. Microbial Ecology of Foods, vols. 1 and 2. Academic Press, New York. Lin, J.K., 1990. Nitrosamines as potential environmental carcinogens in man. Clin Biochem. 23 (1), 67–71. Lueck, E., 1980. Antimicrobial Food Additives: Characteristics, Uses. Effects Springer, Berlin. Norman, N., 1995. Food Science, third ed. AVI Publishing, Westport. Seymour, R.S., Block, S.S. (Eds.), 1983. Disinfection, Sterilisation and Preservation. Lea & Febiger, Philadelphia. Thorne, S., 1986. The History of Food Preservation. Parthenon, Carnforth.
Contents Classification and Properties Permitted Preservatives – Benzoic Acid Permitted Preservatives – Hydroxybenzoic Acid Permitted Preservatives – Natamycin Permitted Preservatives – Nitrites and Nitrates Permitted Preservatives – Propionic Acid Permitted Preservatives – Sorbic Acid Permitted Preservatives – Sulfur Dioxide Traditional Preservatives – Oils and Spices Traditional Preservatives – Organic Acids Traditional Preservatives – Sodium Chloride Traditional Preservatives – Vegetable Oils Traditional Preservatives – Wood Smoke
Classification and Properties M Surekha and SM Reddy, Kakatiya University, Warangal, India Ó 2014 Elsevier Ltd. All rights reserved.
Introduction Fresh foods always contain microorganisms both on their surfaces and within. These microorganisms, if they are not destroyed, will spoil the food. The prevention of food spoilage by inhibiting or destroying the microorganisms is the basis of food preservation. This can be done by chemical treatment, freezing, curing, dehydration, or thermal processing. The chemicals used to prevent food spoilage have some antiseptic properties under the conditions of use and are known as preservatives. Broadly speaking, a preservative is a chemical substance capable of retarding or arresting the growth of microorganisms to prevent such processes as fermentation, acidification, or decomposition, which cause deterioration of flavor, color, texture, appearance, and nutritive value. The main objectives of using preservatives are to extend the shelf life, retain nutritive value, and ensure safety. Chemical preservatives often are used in combination with physical methods; such combinations may allow the preservatives to be used at lower concentrations, thus retaining the quality of the product.
The Need for Preservatives The twentieth century witnessed radical technological advancement in the physical methods of food preservation.
Encyclopedia of Food Microbiology, Volume 3
These developments include preservation of food by thermal processing, refrigeration, freezing, concentration, drying, and more recently the use of irradiation. In spite of this technological advancement, the worldwide population explosion has resulted in a crisis of food supply, which demands a reduction in losses to the minimum. The countries with the greatest nutritional need are the least developed, suffering from inadequate production, distribution, transportation, storage, and preservation facilities. These countries are not in a position to afford the latest technologies for the preservation of food by physical methods, and thus they depend on the use of chemical preservatives that are not only effective but also safe and inexpensive. As physical methods are not suitable for all types of foods, these days even industrial countries are making use of chemical preservatives.
Properties of Preservatives The desirable properties of a chemical substance to serve as a preservative are as follows: 1. A preservative used for antimicrobial purposes should kill the microorganisms rather than inhibit their growth. 2. Any bacteriostatic preservative is most effective if it persists until the food is ready for consumption. If the food is undergoing processing, the bacteriostatic preservative should persist until the food is further processed.
http://dx.doi.org/10.1016/B978-0-12-384730-0.00257-3
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3. A preservative should have an adequate degree of resistance to heat. 4. The specificity range of a preservative should correspond with the range of microorganisms that contaminate and develop on the food. 5. A preservative that is intended to supplant thermal processing should provide a degree of security against Clostridium botulinum similar to that given by the normal thermal processing. 6. The preservative should neither be destroyed by the miscellaneous reactions of the food nor be inactivated by the metabolic products produced by the microorganism. 7. Any antimicrobial preservative should not readily stimulate the appearance of resistant strains of microorganisms. 8. There should be a suitable procedure for determining the amount of the preservative in different foods.
Table 1
Preservatives used in food
Traditional preservatives Sugar Salt Smoke Spices Vinegar Alcohol
Other desirable properties of a preservative are as follows: l
It should have a practical value and be economical. It should be a nonirritant and have low (or no) toxicity. l It should not retard the activity of digestive enzymes or harm the consumer. l Within the body, it should not decompose into substances more toxic than the preservative itself. l
Synthetic preservatives
Bacteriocins
Organic Acetic acid, acetates, and diacetates Sorbic acid and its salts Benzoic acid and its salts p-hydroxybenzoic acid esters and their salts Boric acid and borates Citric acid and its salts Formic acid and formates Lactic acid and its salts Propionic acid and its salts Inorganic Polyamino acids Carbonic acid (CO2) Sulfurous acid and sulfites (SO2) Nitrites and nitrates Phosphates Hydrogen peroxide
Nisin
Bacteriocins
Classification Preservatives include traditional (natural) preservatives, bacteriocins, and synthetic preservatives.
Traditional Preservatives Compounds such as sugar, salt, vinegar, organic fruit acids, wood smoke, alcohol, and various spices used in the preservation of food for centuries are regarded as traditional preservatives. Salts and sugars dissolve in the water of the food to form strong solutions in the process of curing and conserving. The difference between the concentration of the solution and that of the microbial cell cytoplasm causes dehydration of the cell, which leads to its inhibition or death. Salamis, hams, jams, and condensed and sweetened milk are examples of this principle. Smoking destroys bacteria on the surface of food.
Synthetic Preservatives Apart from vinegar, some other acids and their salts are legally permitted preservatives (Table 1). The other synthetic preservatives used are nitrites and nitrates, sulfur dioxide, and sulfites, carbon dioxide, phosphates, and hydrogen peroxide. Chemical preservatives are classified based on their chemical nature and action. On the basis of their chemical nature, they are of two types: inorganic preservatives and organic preservatives. Nitrates, nitrites, sulfites, sulfurous acid, borates, hypochlorites, and peroxide are inorganic preservatives. Benzoates, formic acid, sorbic acid, and propionic acid – and their sodium and calcium salts – as well as esters of p-hydroxybenzoic acid are classified as organic preservatives.
Bacteriocins are a group of small antimicrobial peptides and mostly are plasmid mediated. They generally inhibit only closely related bacteria. Species and strains of Gram-positive lactic acid bacteria (LAB) possess the capacity to produce bacteriocins or bacteriocin-like compounds. Bacteriocins have attracted particular attention as their producer organisms have GRAS (generally recognized as safe) status and are naturally present in many food products. Bacteriocins are a heterogenic group of peptides and can be grouped into the following three classes: 1. Lantibiotics (with 19–37 amino acids), heat stable-Nisin, Lactocin S, Lacticin 3147, and Subtilin 2. Nonlantibiotics (<15 kDa), small and heat stable-Pediocin PA-1, Lactacin B, Lactacin F, Leucocin A-UAL 187, and Lactococcin G 3. Small, heat labile proteins of more than 30 kDa – Caseicin 80, Lacticins A and B Out of many bacteriocins, Nisin is the only purified bacteriocin extensively used as food preservative in many countries. Nisin is a polypeptide with molecular weight 3500 Da, with its rare amino acids (Lanthionine, 3-methyl-lanthionine, dehydroalanine, and dehydrobutyrine). Nisin has several advantages as a food preservative as it is nontoxic, easily degraded by digestive enzymes, thermostable, and does not contribute offflavors and off-odors. Nisin initially forms a complex with a lipid precursor molecule in the formation of bacterial cell walls. The Nisinlipid complex-II then inserts itself into cytoplasmic effuse of essential cellular components, resulting in inhibition or death of bacteria. Gram-negative bacteria are resistant to Nisin because their outer membrane, which is making cell walls, is far less permeable than those of Gram-positive bacteria.
PRESERVATIVES j Classification and Properties Table 2
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Inhibitory action of sorbic acid, benzoic acid, and sulfur dioxide on bacteria, yeasts, and molds Preservatives Sorbic acid
Organism Bacteria Escherichia coli Serratia marcescens Bacillus sp. Clostridium sp. Salmonella sp. Lactobacillus sp. Pseudomonas sp. Streptococcus sp. Micrococcus sp. Yeasts Saccharomyces sp. Hansenula anomala Torulopsis sp. Candida krusei Candida lipolytica Byssochlamys fulva Molds Rhizopus Geotrichum candidum Oospora lactis Penicillium sp. Aspergillus sp. Fusarium sp. a
Benzoic acid a
SO2 a
pH
MICa
pH
MIC
pH
MIC
5.2–5.6 6.4 5.5–6.3 6.7–6.8 5.0–5.3 4.3–6.0
50–100 50 50–1000 100–1000 50–1000 200–700
5.2–5.6
50–120
100–200 50
4.3–6.0 6.0 5.5–5.6 5.2–5.6
300–1800 200–400 50–100 200–400
100
3.2–5.7 5.0 4.6 3.4 5.0 3.5
30–100 500 400 100 100 50–250
3.6 4.8 3.5–4.5 3.5–5.7 3.3–5.7 3.0
120
5.0
25–200 20–100 20–100 100
2.6–5.0 3.0–5.0
200–300 200–500 300–700
30–120 1000 300 30–280 20–300
4.0 5.0
80–160 240
5.0 4.5
160–400 220
MIC, minimum inhibitory concentration, expressed in parts per million (ppm).
Antimicrobial Properties Spectrum of Activity These preservatives do not have a complete spectrum of action against all microorganisms that spoil foods. Most preservatives predominantly act against yeasts and molds (Table 2). In general, most of the organic acids have the broadest spectrum of antimicrobial activity and are useful against many spoilage bacteria, fungi, and yeasts. Benzoic acid is used primarily as an antimycotic agent and most yeasts and molds are inhibited. The activity of benzoic acid against bacteria is variable. Propionic acid and its salts are highly effective mold inhibitors, but yeasts and most bacteria are less affected. Inhibition of ropeforming bacteria in bread is a specific target for propionic acid. Acetic acid is more effective against yeasts and bacteria than molds; Acetobacter sp., certain LAB, and some yeasts are resistant to acetic acid. Lactic and citric acids have only moderate antimicrobial activity. These acids inhibit the formation of aflatoxin and sterigmatocystin. Sorbic acid and its salts have a wide spectrum of activity against catalase-positive bacteria, yeasts, and molds and are highly active against osmophilic yeasts. Sulfur dioxide and sulfites also have a broad spectrum of antimicrobial activity in acid foods. This preservative is more effective against bacteria than molds and yeasts, with Grampositive bacteria being less susceptible than Gram-negative bacteria. Sulfites inhibit enterobacteria and Salmonella. Lactobacilli are highly sensitive to SO2. Yeasts react differently to SO2 depending on the strain. The practical importance of nitrite is in the inhibition of spore-forming bacteria; it also affects
Achromobacter, Aerobacter, Escherichia, Flavobacterium, Micrococcus, and Pseudomonas.
Mechanism of Antimicrobial Action Food preservatives inhibit not only the general metabolism but also the growth of the microorganisms. Depending on the type of preservative used, the final state at which the microorganisms are killed is reached within a few days or weeks, at the usual applied concentrations. The timescale for the killing of microorganisms under the influence of preservatives corresponds to the relationship K ¼ 1=t$ln Z0 =Zt or Zt ¼ Z0 $eKt where K is the death rate constant, t1 is the time period, Z0 is the number of living cells at the time when the preservative begins to act, and Zt is the number of living cells after time t. The given formula is considered to be the basis for studying the action of preservatives in foods. This rule is valid, however, only for relatively high dosages of preservatives and a genetically uniform cell material. A preservative added to a food when microbial counts are low inhibits microorganisms in the initial lag phase; the dosage of preservatives necessary in practice to inhibit microorganisms in the exponential log phase would be too high. Preservatives are not designed to kill microorganisms in substrates already supporting a massive
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PRESERVATIVES j Classification and Properties
germ population. In general, the action of preservatives includes physical as well as physicochemical mechanisms, especially the inhibitory action on enzymes. The partial dissociation of weakly lipophilic acid food preservatives plays an important role in the inhibition of microbial growth. The undissociated lipophilic acid molecules are capable of moving freely through the membrane. They pass from an external environment of low pH (where the equilibrium favors the undissociated molecules) to the cytoplasm, which is of high pH (where the equilibrium favors the dissociated molecules). At the high pH level, the acid ionizes to produce protons, which in turn acidify the cytoplasm and break down the pH component of the proton motive force. To maintain the internal pH, the cell then tries to expel the protons entering it. In doing so, it diverts the energy from growthrelated functions and hence both the growth rate and yield of the cell fall. If the external pH is low and the extracellular concentration of the acid is high, then the cytoplasmic pH drops to a level at which growth is no longer possible and the cell eventually dies. Some preservatives also exert specific effects on metabolic enzymes. Sorbic acid is reported to react with the sulfhydryl groups of enzymes, such as fumarase, aspartase, succinic dehydrogenase, catalase, and peroxidases in bacteria, molds, and yeasts. Antimicrobial activity of organic acids increases with chain length, but the limited water solubility of long-chain acids restricts their use. Benzoic acid is effective only in acid foods. It inhibits enzymes of acetic acid metabolism, oxidative phosphorylation, amino acid uptake, and various stages in the tricarboxylic acid cycle. It also alters membrane permeability of the microbial cell. Transport inhibition is the primary mode of action of parabens. Respiration of microbial cells also is inhibited. Antimicrobial action of propionic acid is due to inhibition of nutrient transport and growth by competing with substances like alanine and other amino acids required by microorganisms. Antimicrobial action of formic acid is similar to any acidulant. Additionally, formic acid inhibits decarboxylase and heme enzymes, especially catalase. The antimicrobial effect of other acids (e.g., lactic, tartaric, phosphoric, and succinic acids) is due to acidification of the microbial cell and inhibiting nutrient transport. Sulfur dioxide is highly reactive, and therefore it interacts with many cell components. The sulfite ion acts as a powerful nucleophile, cleaving the disulfide bonds of proteins, which changes the molecular configuration of enzymes, thus modifying active sites. It reacts with coenzymes (nicotinamide adenine dinucleotide (NADþ)), cofactors, and prosthetic groups such as flavin, thiamin, heme, folic acid, and pyridoxyl. In the case of yeast, the blocking of the oxidation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate is the salient feature. Sulfite treatment of yeast cells results in a rapid decrease in adenosine triphosphate (ATP) content prior to cell death. This is attributed to inactivation of the enzyme glyceraldehyde-3-phosphate dehydrogenase. Sulfite also reacts with carbonyl constituents of the metabolic pool to form hydroxysulfonates. Yeasts when treated with sublethal concentrations of sulfite tend to excrete increased amounts of acetaldehyde. This is due to the trapping of this metabolic intermediate as the stable hydroxysulfonate, thereby preventing its conversion to ethanol so that the reaction equilibrium shifts. Glycerol is
formed instead of ethanol by reduction of glyceraldehyde-3phosphate to glycerol-3-phosphate, which subsequently is dephosphorylated. In Escherichia coli NAD-dependent formation of oxalacetate from malate is inhibited. Sulfite destroys the activity of thiamin by breaking the bond between the pyrimidine and thiazole portion of the molecule. The antimicrobial action of nitrite is based mainly on the release of nitrous acid and oxides of nitrogen. Nitrite inhibits active transport of proline in E. coli and aldolase from E. coli, Enterococcus faecalis, and Pseudomonas aeruginosa. Reaction between nitric oxide from the nitrite and iron of a cidophore compound involved in electron transport in clostridia accounts for the anticlostridial action. Nitrite reacts with heme proteins such as cytochromes and sulfhydryl enzymes, resulting in the formation of S-nitroso products.
Combination of Preservatives No single preservative is active against all spoilage microorganisms. Attempts have been made to compensate for this by combining various preservatives with different spectra of action. In general, organic acids are compatible with other preservatives and many combinations are synergistic, for example, the following: l
Benzoate with SO2, CO2, NaCl, boric acid, or sucrose Propionate with CO2 or sorbate l Sorbate with sucrose or NaCl l Lactic acid with acetic acid. l
The combinations of sorbic acid, benzoic acid, or esters of phydroxybenzoic acid with Nisin and tylosin are useful because they extend the spectrum of action to cover E. coli, Lactobacillus, and Staphylococcus strains. Cured meats are rarely involved in Clostridium perfringens food poisoning. This is a fine example of the hurdle concept: Individual preservatives such as salt content, nitrite, and heat processing are insufficient to ensure safety but effectively control growth of C. perfringens in combination. Contrary to general expectations, not all combinations give better results than individual constituents. The presence of one preservative may sometimes weaken the effect of the other. For instance, boric acid has the tendency to weaken the effect of other preservatives in their action on E. coli. Its action against fungi proved to be synergistic, however. On the other hand, the presence of some chemical substances such as calcium chloride, which is not a food preservative and has no antimicrobial effect individually, slightly weakens the efficacy of sorbic acid, benzoic acid, and other preservatives. In general, a beneficial effect will be obtained by using preservatives with substances that counter dissociation, such as acids, or those that reduce water activity, for example, NaCl or sugar.
Degradation of Preservatives In general, food preservatives are stable substances and are unlikely to decompose within the specified storage time. Occasionally, however, certain preservatives such as organic compounds are decomposed by microorganisms and are used as a source of carbon by them. Decomposition of this type of preservative is possible, if the preservative is ineffective against microbes and also if the food contains a large number of
PRESERVATIVES j Classification and Properties microbes. Therefore, it is impossible with such preservatives to arrest the spoilage of food and to maintain the food in an apparently fresh condition. The best example of this phenomenon is the conversion of sorbic acid to hexadienol by some strains of LAB. This product reacts with ethanol to form 1ethoxy-2,4-hexadiene and 2-ethoxy-3,5-hexadiene, which give a geranium-type odor in wines.
Interaction of Preservative with Food Components Chemical reaction between food preservatives, food components, and microorganisms may lead to the formation of reaction products of toxicological importance and reduction in the concentration and the activity of the preservative. Some food preservatives such as sorbic acid, SO2, sulfites, and nitrites have an extensive reactivity with food components. Sorbic acid reacts with low-molecular-weight thiols of food, such as cysteine and glutathione, to form the 5-substituted 3hexenoic acid. Sorbic acid also undergoes autooxidation to malonaldehyde, acetaldehyde, and b-carboxyacrolein. Owing to its high chemical reactivity, sulfur dioxide may be involved in a variety of interactions with food ingredients. The action of SO2 in destroying thiamin in food is significant. An important nucleophilic reaction of the sulfite ion is its addition to the a-b-unsaturated carbonyl moiety of 3,4-dideoxyosulos3-enes formed as reactive intermediates in Maillard and ascorbic acid browning, which causes a considerable depletion of the preservatives in foods susceptible to nonenzymatic browning. The nitrite added to meat is converted to nitric oxide, which combines with myoglobin to form nitric oxide myoglobin. The N-nitrosamines formed by the cooking of nitrite-cured meat are potent carcinogens. Nitrosophenols formed by C-nitrosation of phenolic components of food are readily oxidized to the corresponding nitro compounds; S-nitroso compounds are readily formed by the nitrosation of thiols and represent a reversibly bound form of the preservative.
Uses Preservatives applicable to a particular need are determined by the composition of the food, the type of microbial spoilage, and the desired shelf life. As well as the specific physical properties, cost is also an important consideration in the selection of preservative. Preservatives are incorporated directly into food products or developed during processing food. Traditional preservatives have been introduced through processes such as fermentation, salting, curing, and smoking. Spices are commonly added in small amounts to the food as a preservative. Sugar is used in the preservation of jams, jellies, candied fruit, and sweetened condensed milk. The main use of smoke is to preserve meat and fish products. Salt is used to preserve many foods, including butter, margarine, cheese, sausages, ham, and fish. Chemical preservatives may be applied directly, most often as an ingredient of manufactured foods, but also by dipping, spraying, gassing, or dusting. Some preservatives are incorporated in the packing material rather than applied directly to the food. Vinegar or acetic acid is used in many foods, including
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mayonnaise, catsup, salad dressings, pickles, and meat. Lactic acid is used as a flavoring agent in frozen desserts and as an emulsifier in bakery products. It is also used for faster nitrite depletion and botulinal protection due to lowered pH in meat product processing. Sodium benzoate is the most widely used preservative for acid foods, including carbonated and still beverages, salads, fruit desserts, fruit cocktails, and margarine. Sodium benzoate is used at concentrations of 0.03–0.10%. Because of the astringent flavor of benzoates, they often are used in combination with sorbate or parabens. Parabens are used to preserve soft drinks, fruit products, jams, jellies, pickles, cream, and pastes. The N-heptyl ester can be used in beer fermentation at a level of 12 mg g1. Parabens may be added as a dry or liquid ingredient to food. Sorbic acid and its salts frequently are used because of their high solubility. Sorbates are used in cheese products, baked foods, fruits, fruit juices, vegetables, soft drinks, wines, jellies, jams, syrups, salads, margarine, and fish products. Since sorbate inhibits yeasts, it is not used in yeast-raised bread. Sorbate may be added directly to the food or it may be applied by dipping, spraying, dusting, or impregnating packing materials and wrappers. Recent studies showed the effectiveness of sorbate as an antibotulinal agent in meat products. In food processing, gases such as SO2 and CO2 may be used as antimicrobial agents or for other purposes. These gases may have direct or indirect antimicrobial effects. Sulfur dioxide is principally used in wine preservation. It is employed as a liquid under pressure or in aqueous solution. Additionally, various sulfite salts (sodium sulfite, sodium hydrogen sulfite, sodium metabisulfite, potassium metabisulfite, and calcium sulfite) containing 52–68% active SO2 are used to preserve a variety of foods, such as fruit juices, soft drinks, dehydrated fruits and vegetables, pickles, syrups, meat, and fish products. In wine, sulfite also is used as an equipment sanitizer, antioxidant, and clarifier and to prevent bacterial spoilage during storage. A combination of 200 mg sorbic acid, 220 mg potassium sorbate, and 20–40 mg SO2 per liter provides a comprehensive protection to the wine. Proteolytic breakdown of meat may be prevented by sulfites. Sulfur dioxide is added to foods to prevent enzymatic reactions, notably browning. Carbon dioxide is used to control psychrotrophic spoilage of meat and meat products, poultry, fish, eggs, fruits, and vegetables. Carbon dioxide generally is used in the form of a liquefied gas or as dry ice (solid CO2), which sublimes to form CO2 gas. Carbon dioxide applied under pressure with low temperature results in rapid biocidal action. Carbon dioxide is a major applicant in carbonized soft drinks, mineral water, wines, beers, and ales. It functions as an antimicrobial and effervescing agent. It inhibits aerobic spoilage organisms when used in vacuum-packed meats at a concentration of 10–20%; higher concentrations may cause undesirable odors. The combination of O2 and CO2 in a controlled atmosphere delays the respiration, ripening, and spoilage of stored fruits and vegetables. Nitrites are added to cheese and meat products. The addition of nitrite not only prevents the growth of toxigenic microorganisms but also the production of toxins. Nitrite added to meat results in both chemical and antimicrobial effects. It reacts with heme proteins to form the characteristic
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PRESERVATIVES j Classification and Properties
cured meat color and has a mild antioxidant effect that prevents rancidity and a warmed-over flavor. At low pH, nitrite is depleted by increased formation of nitrous acid and nitric oxides, which are the reactive forms of nitrite. Because of this, the addition of acids, acidulant, or glucono-delta-lactone has a beneficial effect on the action of nitrite. For the positive chemical effect (color and flavor), a nitrite concentration of 50 mg g1 is needed, whereas antibotulinal activity requires a concentration of 100 mg g1. Lower concentrations of nitrite (40–80 mg g1) in combination with sorbate are more effective. In the United States, the content of sodium nitrite in cured meat products is limited to 200 mg g1, with specific regulatory levels varying with the product. In the United Kingdom, potassium and sodium nitrite are permitted in cured meats up to a maximum of 200 mg g1. Nisin has been used to preserve dairy products, egg products, pasteurized soups, flavor-based products, canned foods, meat products, sea foods, salad dressing, and alcohol beverages. Nisin is predominantly sporostatic rather than sporicidal and, for this reason, it is widely used as a natural preservative. Ethylenediaminetetraacetic acid enhances the antimicrobial activity of Nisin against Gram-negative bacteria. Nisin is stable at pH 2.0 and can be autoclaved at 121 C. Increasing alkalinity results in the loss of antimicrobial activity of Nisin. Nisin is used in canned products as a sterilizing auxiliary. Natamycin (pimaricin) is permitted in some western European countries for surface preservation of cheese and as an additive to cheese coating. It has been used to retard yeast and mold spoilage of fruit, fruit juices, cottage cheese, poultry products, and sausage.
Toxicology and Regulatory Status Traditionally processed food in general finds ready acceptance by regulatory authorities. This is not the case for foods processed by the addition of chemical preservatives, where it is essential to ensure that the preservative used does not become a health hazard to human beings. Benzoic acid and its salts have low toxicity in experimental animals and humans. Humans have a high tolerance to sodium benzoate because of a detoxifying mechanism, in which benzoate and glycine or glycuronic acid are conjugated and excreted as hippuric acid or benzoyl glucuronide. Benzoate is not mutagenic in Drosophila or Salmonella but interacts with nucleosides and DNA in vitro. Sodium benzoate and benzoic acid are GRAS at concentrations up to 0.1% in the United States. In the United Kingdom, benzoic acid and its salts are permitted on a wide scale in accordance with the Preservatives in Food Regulations of 1979. Parabens toxicity is low, with an acute toxicity dose LD50 of 180–8000 mg kg1 of body weight in experimental animals, varying with the form of administration. The acceptable daily intake (ADI) is 10 mg kg1 of body weight of average human. The methyl and propyl parabens are GRAS in the United States, with a total addition limit of 0.1%. Propionic acid and its salts are readily absorbed by the digestive tract owing to their high water solubility. This acid decomposes in mammals by linkage with coenzyme A via methylmalonyl-CoA, succinyl-CoA, and succinate to yield CO2 and H2O. The ADI set by the Food and Agriculture
Organization (FAO) and the World Health Organization (WHO) is not limited. These preservatives are permitted for use in many countries. Sorbic acid is nontoxic and is metabolized by fatty acid oxidation, pathways common to both laboratory mammals and humans. The oral LD50 for rats is 7–10 g kg1 of body weight, and 6–7 g kg1 of body weight for the sodium salt. In highly sensitive individuals, this preservative irritates the mucous membranes. Sorbates have no mutagenic, teratogenic, or carcinogenic action. The FAO/WHO acceptable daily intake of sorbic acid and its salts is 25 mg kg1 of body weight, the highest ADI of the common preservatives. In the United States, sorbic acid and sorbates are GRAS. The maximum permissible level is 0.1–0.2%. These compounds are permitted in all countries for preservation of a wide variety of foods. Sulfur dioxide and sulfites in the body are oxidized to sulfate and excreted in urine. Although vitamin B1 deficiency, diarrhea, organ damage, and decreased usage of dietary protein and fat are some of the adverse effects of SO2 in human beings, actual poisoning by SO2 and sulfite is not possible because of vomiting. Sulfite is not a carcinogen, but SO2 is mutagenic. Levels of application are restricted to 500 mg g1 owing to flavor problems. The FAO/WHO acceptable daily intake of SO2 and sulfites is 0.7 mg kg1 of body weight per day for the average human. It is difficult to estimate an average human intake of SO2 since consumption of treated foods is high. Sulfur dioxide intake may sometimes exceed the ADI value. For example, the consumption of about three glasses of wine per day alone leads to an SO2 intake exceeding the ADI. Sulfur dioxide destroys the thiamin in foodstuffs, and many of the reported toxicity problems are symptoms of thiamin deficiency. It also interacts with folic acid, vitamin K, and certain flavins and flavoenzymes. This problem can be overcome by supplementing nutritionally adequate diet, which can withstand substantial intakes of SO2 in terms of thiamin destruction. Humans ingesting up to 200 mg SO2 per day showed no signs of thiamin deficiency or changes in urinary excretion. Products of nitrite, which reduces hemoglobin and increases the methaemoglobin content of the blood, are highly toxic to humans. Methaemoglobinemia may result in death due to oxygen shortage. Infants less than 6 months old are particularly susceptible. Neither nitrate nor nitrite have teratogenic action. The formation of potent carcinogenic compounds, nitrosamines, in cooked cured meat products can be reduced by the combination of nitrite with other preservatives, such as sorbic acid or common salt. The LD50 of nitrite for human beings is 300 mg kg1 body weight. Sodium and potassium nitrite are permitted in many countries, including the United States and the United Kingdom, to preserve meat and fish products and cheese. Nitrate contributes little or no preservative action except as a source of nitrite (e.g., following reduction by Micrococcus species in curing or fermenting meats). The FAO and WHO acceptable daily intake of nitrate is 0–5 mg kg1 per day and for nitrite 0–0.2 mg kg1 per day.
See also: Clostridium: Clostridium botulinum; Preservatives: Traditional Preservatives – Oils and Spices; Traditional
PRESERVATIVES j Classification and Properties
Preservatives: Sodium Chloride; Preservatives: Traditional Preservatives – Organic Acids; Preservatives: Traditional Preservatives – Wood Smoke; Permitted Preservatives: Sulfur Dioxide; Preservatives: Permitted Preservatives – Benzoic Acid; Permitted Preservatives – Hydroxybenzoic Acid; Permitted Preservatives: Nitrites and Nitrates; Preservatives: Permitted Preservatives – Sorbic Acid; Preservatives: Permitted Preservatives – Nisin; Permitted Preservatives – Propionic Acid; Preservatives: Traditional Preservatives – Vegetable Oils.
Further Reading Adams, M.R., Moss, M.O., 1996. Food Microbiology. New Age International, New Delhi. Branen, A.L., Davidson, P.M. (Eds.), 1983. Antimicrobials in Food. Marcel Dekker, New York.
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Branen, A.L., Davidson, P.M., Salminen, S. (Eds.), 1990. Food Additives. Marcel Dekker, New York. Busta, F.F., Foegeding, P.M., 1983. Chemical food preservatives. In: Block, S.S. (Ed.), Disinfection, Sterilization and Preservation, third ed. Lea and Febiger, Philadelphia, pp. 656–694. Davidson, P.M., Branen, A.L. (Eds.), 1993. Antimicrobials in Foods, second ed. Marcel Dekker, New York. Gould, G.W. (Ed.), 1989. Mechanism of Action of Food Preservation Procedures. Elsevier, London. Hayes, P.R., 1985. Food Microbiology and Hygiene. Elsevier, London. ICMS, 1980. Microbial Ecology of Foods, vols. 1 and 2. Academic Press, New York. Lin, J.K., 1990. Nitrosamines as potential environmental carcinogens in man. Clin Biochem. 23 (1), 67–71. Lueck, E., 1980. Antimicrobial Food Additives: Characteristics, Uses. Effects Springer, Berlin. Norman, N., 1995. Food Science, third ed. AVI Publishing, Westport. Seymour, R.S., Block, S.S. (Eds.), 1983. Disinfection, Sterilisation and Preservation. Lea & Febiger, Philadelphia. Thorne, S., 1986. The History of Food Preservation. Parthenon, Carnforth.