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In the United States, benzoic acid and sodium benzoate are GRAS preservatives (Code of Federal Regulations, 1977/1988; Title 21, Secs. 184.1021 and 184.1733) up to a maximum permitted level of 0.1%. They may also be used in certain standardized foods. In most other countries, the maximum permissible quantities generally range between 0.15% and 0.25%. They may also include foods from which benzoate is excluded in the United States. An ADI (average daily intake) value of 0 to 5 mg/kg of body weight has been specified for benzoic acid (Pollard, 1990).

A Food Technology special report (1986) reviewed the characteristics, applications, and limi-tations of preservatives and antimicrobial agents, including benzoic acid. Potassium benzoate is now commercially available and apparently evolved in response to consumers’ interest in reduced sodium intake. Regulatory agencies consider it to be GRAS to the same extent that sodium benzoate is GRAS as a preservative with the limitation of 0.1%. The potassium salt is less water soluble (42.4 g per 100 ml solution) than the sodium salt. Approximately 1.1 times more potassium salt than sodium salt is required to obtain the same level of benzoic acid in solution and the same level of antimicrobial effect. Product information for both salts is available (Miles Laboratories, Inc.).

Calcium benzoate has also been approved for use, although its solubility in water is much less than the sodium salt (Pollard, 1990).

APPLICATIONS

Benzoic acid and sodium benzoate are most suitable for foods and beverages that naturally are in a pH range below 4.5 or that can be brought into this range by acidification. As food preservatives, their main advantages include low price, ease of incorporation into products, and lack of color.

However, the narrow pH range in which they are effective, the off-flavor they may impart to the foods they are to preserve, and their toxicologic properties compared to some other preservatives have all contributed to recent efforts by food processors to replace benzoic acid and sodium benzoate with other preservatives with better characteristics. Benzoate does not control the growth of high levels of microorganisms and therefore cannot be used to compensate for poor ingredients or processing.

USEIN VARIOUS FOOD SYSTEMS

Benzoic acid and sodium benzoate have been widely used to preserve beverages, fruit products, bakery products, and other foods in several countries (Tables 2.8 and 2.9). Applications of these preservatives to foods have been reviewed by Chichester and Tanner (1972), Lueck (1980), Kimble (1977), and Chipley (1983, 1993). In addition, the use of benzoic acid and other chemicals in the processing and freezing of shrimp has been reviewed (Chandrasekaran, 1994).

Recent reports indicate that these preservatives continue to be of benefit in a variety of products.

Fresh catfish were dipped in various concentrations of sodium benzoate or potassium sorbate and smoked, then evaluated during shelf life (Efiuvwevwere and Ajiboye, 1996). Sorbate treatment was more effective than benzoate and increased the shelf life by 8 days.

Halotolerant fungi were isolated from salted dried fish (Chakrabarti and Varma, 2000). Aspergil-lus flavus, A. niger, and Penicillium sp. were dominant. Penicillium sp. was most sensitive to propionic acid and sodium benzoate, but A. flavus was most sensitive to potassium sorbate. Propionic acid (0.06%) or potassium sorbate (0.02%) or sodium benzoate (0.04%) inhibited growth of all three fungi.

Several additives and preservatives were evaluated as inhibitors of melanosis (black spot) and microbial spoilage in prawns in chilled storage (Montero et al., 2001). A combination of sodium benzoate and kojic acid was effective in inhibiting melanosis. However, 4-hexylresorcinol was the most effective inhibitor of both melanosis and microbial spoilage in prawns. The shelf life of sardines stored in an acid brine containing 0.3% sodium benzoate was three times longer than that of the controls (Ponce de Leon et al., 1994).

The effectiveness of sodium benzoate and potassium sorbate, separately or combined, was evaluated against the growth of Z. bailii in an inoculated salsa mayonnaise stored at room temper-ature (Wind and Restaino, 1995). Potassium sorbate was more effective than sodium benzoate.

However, neither preservative, separately or combined, prevented spoilage of the product by Z. bailii.

TABLE 2.8

Maximum Permitted Levels (ppm) for Benzoic Acid and Its Salts in Foods in Selected Countries

Food Canada Denmark Norway Sweden U.K. U.S.^a

Fish products 1000

Fish, semipreserved 1000 1000 5000 2000 1000

Fruit juice 1000 200 1500 1000 800 1000

Fruit pulp 1000 1500 1000 800 1000

Jam, jellies 1000 500 1500 1000 1000

Liquid egg white, yolk or whole

500 5000 10,000

Margarine, salted 1000 2000 1000

Mayonnaise 1000 3000 2000 1000

Mustard 1000 1000 3000 2000 1000

Pickles, relishes 1000 1000 1500 2000 250 1000

Salads, salad dressings 1000 1000 1500 2000 250 1000

Sauces, ketchup 1000 1000 1500 2000 250 1000

Soft drinks containing fruit juice

1000 200 1500 1000 800^b 1000

Soft drinks, carbonated 1000 200 500 1000 160^c GMP^d

a In the United States, a maximum level of 1000 ppm benzoic acid or its salts may be used for all products listed. A maximum level of 2000 ppm may be used in orange juice for manufacturing. Orange juice not for manufacturing may not contain a preservative (federal standards of identity).

bSoft drinks for consumption after dilution.

c Soft drinks for consumption without dilution.

dGood manufacturing practice.

Source: Adapted from Chipley (1993).

Sodium Benzoate and Benzoic Acid 31

The microbial stability and quality of tomato juice and fermented cucumbers and carrots were improved by addition of a combination of sorbate and benzoate or benzoate alone (Bizri and Wahem, 1994; Fleming et al., 1996; Montano et al., 1997). The levels of some nutrients were reduced when tomato juice was treated with dimethyl dicarbonate. Tomato concentrate could be kept for up to 12 months at room temperature with the addition of salt, acetic acid, sodium benzoate, and potassium sorbate (Uboldi Eiroa et al., 1995). No viable microorganisms, including Z. bailii, were observed in any samples containing preservatives.

The effects of sodium benzoate and potassium sorbate on the thermal death rates of ascospores from the heat-resistant mold N. fischeri were evaluated in fruit juices (Rajashekhara et al., 1998).

Comparable rates were noted when each preservative or the combination of both was used in mango juice heated to 85°C. In grape juice, potassium sorbate was more effective than sodium benzoate or their combination.

Growth and control of four Salmonella serotypes in a soft Hispanic type cheese were evaluated (Kasrazadeh and Genigeorgis, 1994). The minimum temperature that allowed growth was 8°C.

Growth and control of two strains of E. coli O157:H7 were also evaluated in this cheese (Kasrazadeh and Genigeorgis, 1995). The minimum temperature that allowed growth of the E. coli strains was 10°C. In both studies, models were developed relating lag time and specific growth rate to

TABLE 2.9

Maximum Permitted Levels (ppm) for Benzoic Acid and Its Salts in Foods in the European Union^a

Commodity Food Level for Use

Beverages Nonalcoholic flavored drinks (excluding dairy-based drinks) Spirits with <15% alcohol by volume

Alcohol-free beer in keg Fish Semi-preserved fish products (including fish roe)

Salted, dried fish

Low-sugar jams, jellies, marmalades, and other fruit-based spreads Candied fruits and vegetables

Vegetables in vinegar, brine, or oil Prepared salads Sauces Emulsified sauces with fat content ≥60%

Emulsified sauces with fat content <60%

Nonemulsified sauces Liquid egg white, yolk, or whole egg Liquid dietary food supplements

Dietetic foods (excluding foods for infants and young children)

300

a The European Union (EU) is currently composed of 12 member nations. In the EU, benzoic acid and its sodium, potassium, and calcium salts are all permitted for use (E210–E 213) (Pollard, 1990).

Source: Adapted from Anon. (1995).

temperature. Growth was either prevented or delayed by adding sodium benzoate (0.3%) to cheese (pH 6.6) or adding potassium sorbate (0.3%) to cheese (pH 6.0) made from milk acidified to pH 5.9 with propionic acid.

Chicken skin was inoculated with Salmonella spp., L. monocytogenes, C. jejuni, or S. aureus, and wash solutions were evaluated for effectiveness in decontaminating the skin (Hwang and Beuchat, 1995). Washing the skin with solutions of either 0.3% or 0.5% lactic acid combined with 0.05% sodium benzoate reduced the numbers of these pathogens compared to washing with water.

No viable cells from any of the four pathogens were detected on skin washed with lactic acid/ben-zoate solutions and stored for 8 days at 4°C. These solutions could be used for dips to sanitize chickens intended for frying before presentation to consumers (Hathcox et al., 1995).

Listeria monocytogenes is recognized as a foodborne pathogen of major concern to humans.

Large outbreaks of infection have been linked to consumption of contaminated coleslaw, cheeses, milk, hot dogs, and luncheon meats. Contamination of many of these foods frequently occurs after processing. Application of antimicrobials as sprays or mists to meats following processing may be more effective than their addition in the meat formulation. The antimicrobial can be applied directly onto the product surface where cells of L. monocytogenes usually attach following cooking and during slicing and packaging (Farber and Peterkin, 1999; Tompkin et al., 1999). A study was conducted to evaluate aqueous dipping solutions of organic acids or salts to control L. monocyto-genes on sliced, vacuum-packaged bologna stored at 4°C for up to 120 days. No significant increase in the numbers of this pathogen occurred on bologna slices treated with 2.5% or 5% acetic acid, 5% sodium diacetate, or 5% potassium benzoate for 120 days (Samelis et al., 2001). Sorbates and benzoates are currently approved in various countries for use as dipping solutions to prevent fungal growth in dry sausages (Sofos, 1989).

Combinations of organic acids, low pH, and ethanol were very effective bactericidal treatments for L. monocytogenes (Barker and Park, 2001). Benzoate was one of the most effective compounds tested in the presence or absence of ethanol. Ethanol-enhanced killing correlated with damage to the bacterial cytoplasmic membrane.

Unpasteurized apple cider has been implicated in several foodborne disease outbreaks caused by pathogenic microorganisms. Most research efforts have been directed toward E. coli O157:H7 in cider because of the severity of illness this pathogen causes, especially in younger children and the elderly. Survival of E. coli O157:H7 in apple juice and apple cider with or without potassium sorbate or sodium benzoate has been investigated. Both preservatives reduced the heat resistance of E. coli O157:H7, but benzoate was about two to eight times more effective than sorbate (Splittstoesser et al., 1996; Dock et al., 2000). At 8°C, the combination of sorbate and benzoate was more effective than either preservative alone (Zhao et al., 1993). Resistance to these preserva-tives was greater at 4°C than at 25°C (Fisher and Golden, 1998). These strains grew well in unpasteurized and pasteurized apple juice; growth was inhibited by benzoate or sorbate (Koodie and Dhople, 2001). Control strains of E. coli failed to grow in either type of apple juice. However, the growth of some O157:H7 strains in apple cider was not affected by the presence of either preservative (Miller and Kaspar, 1994).

A preservative treatment was developed that was capable of achieving the FDA mandate for a 5-log reduction of E. coli O157:H7 in apple cider (Comes and Beelman, 2002). The treatment that was successful included addition of 0.15% fumaric acid and 0.05% sodium benzoate followed by holding at 25°C for 6 hours and at 4°C for 24 hours. The same preservatives added to cider resulted in a greater than 5-log reduction in less than 5 and 2 hours when held at 25°C and 35°C, respectively.

USEAS A POSTHARVEST FUNGICIDE

Because of the long and successful use of benzoic acid in the processed food industry, this antimicrobial was evaluated for control of postharvest diseases of various fruits and vegetables.

However, several factors may influence the effectiveness of benzoic acid in the treatment of fresh

Sodium Benzoate and Benzoic Acid 33 fruits and vegetables, with perhaps the most pertinent consideration the pH of the superficial tissues (Eckert, 1967).

A benzoic acid-based polymer coating for apples has been developed (Ivanov et al., 1989). A mixture of fruit-coating polymers and potassium sorbate or sodium benzoate completely inhibited postharvest fungal growth on bananas (Al Zaemey et al., 1994). The shelf life of sliced apples and potatoes was extended by 1 week when they were dipped in a polysaccharide/protein edible coating containing sodium benzoate or potassium sorbate (Baldwin et al., 1996). Methylcellulose was mixed with chitosan and 4% sodium benzoate or potassium sorbate to produce a food-grade film (Chen et al., 1996). Significant antifungal properties were demonstrated against food spoilage fungi.

Benzoic acid and its derivatives have been proposed for use as fungicides, especially against toxigenic strains of A. flavus, in peanuts (Uraih and Offonry, 1981). It is currently used in animal feeds and in some tobacco products as a fungicide at levels of up to 0.1% and 0.025%, respectively.

OTHER APPLICATIONS

Benzoic acid is one of the oldest preservatives in the cosmetic and pharmaceutical industries.

Generally, concentrations of 0.1% to 0.5%, incorporated as sodium benzoate, have been used to preserve cosmetic formulations. Currently, the cosmetic applications for benzoic acid have largely been taken over by more potent antimicrobial agents and compounds that are active over a wider pH range (Manowitz, 1968). A summary of the antimicrobial activities of 30 preservatives, including benzoic acid, used in cosmetics has been published (Bach et al., 1990). In the pharmaceutical industry, benzoic acid was used for oral preparations in concentrations of 0.05% to 0.1%. Although its use in pharmaceuticals has largely been displaced by other compounds (Grundy, 1968), possible areas for specialized applications still exist — for example, as a disinfectant for artificial kidneys (Kolmos, 1976), as a control for dentureborne Candida albicans (Lambert and Kolstad, 1986), and as a preservative in certain brands of cough syrup (Chen et al., 1988; Hewala, 1994).

The minimum inhibitory concentrations of sodium benzoate and dichlorobenzyl alcohol for 115 strains of dental plaque microorganisms were determined (Ostergaard, 1994). Sodium benzoate did not inhibit growth of any Gram-positive cocci. However, saliva samples from volunteers who used a dentifrice containing these antimicrobials indicated that for 5 to 10 minutes after toothbrush-ing, their concentrations were high enough to inhibit growth of periodontal pathogens. A single oral rinse with a mouth rinse containing these antimicrobials did not affect plaque removal (Danielsen et al., 1996).

A sodium benzoate–sorbic acid combination used in a pharmaceutical product for treating ulcers was effective against a wild strain of Pseudomonas cepacia, following official methods of analysis (Zani et al., 1997). However, this preservative system was ineffective against an adaptive-resistant strain. A p-hydroxybenzoate-based system was effective in protecting the product against a variety of strains of P. cepacia grown under different conditions.

Sodium benzoate has also been used to inhibit postharvest changes in fruits and vegetables.

For example, Wang and Baker (1979) found that chilling injury to cucumber and sweet pepper fruits could be reduced by addition of 10 mM sodium benzoate as a 5-minute dip before chilling.

The chilling-induced production of ethylene in cucumbers was inhibited by the addition of sodium benzoate (Wang and Adams, 1980).

Patents have also been issued for the use of benzoic acid or sodium benzoate as an industrial fungicide in several products. These include animal feedstuffs; varnishes; modified starch adhesives;

adhesives for paper, textiles, and leather; lubricants; liquid coolants; rubber manufacturing; sealing gaskets for food containers; insoles for shoes; wood preservatives; and treatment for microbial infections in trees.

STORAGEAND HANDLING

Sodium benzoate should be stored in a cool, dry place. Containers should be kept closed as much as possible. This product is not corrosive. There is little danger of its being a toxicant or fire hazard under normal conditions of use. However, both benzoic acid and sodium benzoate are moderately toxic by ingestive, intramuscular, and intraperitoneal routes (Sax and Lewis, 1989).

TOXICOLOGY

Several toxicologic studies involving different animal species have been conducted using both benzoic acid and sodium benzoate. These have been summarized by exposure category in Table 2.10. In addition, Sax and Lewis (1989) reported data for several animal species based on the route of administration (Table 2.11). Extensive human feeding trials conducted early in the twentieth century led Chittenden et al. (1909) and Dakin (1909) to conclude that sodium benzoate was not deleterious to human health.

Benzoate does not appear to be accumulated in the body. The apparent reason for this involves a detoxifying mechanism whereby benzoate is absorbed from the intestine and “activated” by linkage with CoA to yield benzoyl coenzyme A. The overall reaction sequence proceeds as shown in Figure 2.7 (White et al., 1964).

The first reaction is catalyzed by a synthetase enzyme; the second is catalyzed by an acyltrans-ferase enzyme. Hippuric acid, synthesized in the liver, is then excreted in the urine (White et al., 1964). Its formation can be increased greatly by administration of benzoic acid. Excretion of benzoic

TABLE 2.10

Toxicity of Benzoic Acid and Sodium Benzoate by Exposure Category^a

Category

Species

Tested Time Period Level Results

Acute Rat 1.7–4.0 g/kg body weight 50% mortality

Guinea pig,^b rabbit, cat, dog

1.4–2.0 g/kg body weight 100% mortality

Subchronic Mouse 3 months 80 mg/kg body weight Mortality rate increase

Mouse 5 days 3% of diet 50% mortality

Mouse 3 months 4% of diet (as sodium benzoate) No effect

Human 3 months 1 g/day No effect

Human 14 days 12 g per 14 days No effect

Human 60–100 days 0.3–4.0 g per 60–100 days No effect

Human Several days 5–10 g for several days (as sodium benzoate)

No effect

Chronic Mouse 17 months 40 mg/kg body weight per day Growth disturbance Rat 18 months 40 mg/kg body weight per day Growth disturbance

Rat 2 weeks 5% of diet (as sodium benzoate) 100% mortality

Rat 1.5% of diet Decreased growth rate

Rat 2 weeks 1% of diet (as sodium benzoate) No effect

a Route of administration was peroral.

bRoute of administration was per os.

Source: Adapted from Lueck (1980).

Sodium Benzoate and Benzoic Acid35

TABLE 2.11

Toxicity of Benzoic Acid and Sodium Benzoate by Route of Administration

Benzoic Acid Sodium Benzoate

a Scu, subcutaneous; Ipr, intraperitoneal; Ims, intramuscular.

bTDLo, lowest published toxic dose; LDLo, lowest published lethal dose; LD50, lethal dose 50 percent kill; MLD, mild irritation effects; SEV, severe irritation effects.

c Total dosage.

dTotal dosage for 22 days to pregnant rats.

e Total dosage for 3 days to pregnant rats.

Source: Adapted from Sax and Lewis (1989).

acid by this particular pathway was first reported by Dakin (1909). Griffith (1929) found that this mechanism accounted for 66% to 95% of benzoic acid fed in studies in which the quantities were far in excess of those that might be ingested from foods preserved with permitted levels of benzoate.

He also suggested that the remaining portion of the benzoate not excreted as hippuric acid may have been detoxified by conjugation with glucuronic acid and excreted in the urine by this route.

Akira et al. (1994) used deuterated benzoic acid and nuclear magnetic resonance spectroscopy to monitor metabolism in a healthy male volunteer. A single oral dose of 250 mg of benzoic acid was quantitatively metabolized to hippuric acid and excreted in the urine within 4 hours. The amount of sodium benzoate absorbed by human subjects may be estimated by determining the amount of hippuric acid excreted in the urine (Fujii et al., 1991).

Since the late 1970s, benzoic acid has been used in the treatment of patients with congenital errors of urea synthesis to develop an alternative pathway of nitrogen waste excretion (Kubota and Ishizaki, 1991). These authors analyzed plasma concentrations of benzoic and hippuric acids and urinary levels of hippuric acid after oral doses of sodium benzoate (40, 80, or 160 mg/kg) were given at least 1 week apart to 6 healthy male volunteers. The authors reported that both the biotransformation of benzoic acid to hippuric acid and the urinary excretion of hippuric acid followed first-order reaction rates.

These results have been confirmed in several animal studies. Different animal species differ in their ability to metabolize benzoic acid. This may be related to differences in the ability of liver and kidney cells to carry out glycine and glucuronic acid conjugation (for review, see Chipley, 1993).

Sodium benzoate inhibited the synthesis of glucose from lactate and generation of urea from ammonia when added to suspensions of rat hepatocytes (Cyr et al., 1991). Inhibition was caused by accumulation of benzoyl CoA with a resultant depletion of free CoA and acetyl CoA. Accel-eration of the conversion of benzoyl CoA to hippurate by the addition of glycine restored the levels of free CoA and acetyl CoA and the rates of gluconeogenesis and ureagenesis.

In guinea pigs, the route of administration significantly affected the efficiency of benzoic acid metabolism (Nathan et al., 1990). When applied topically, only a small portion (6.9%) of the absorbed benzoic acid was conjugated with glycine to form hippuric acid. However, it was excreted almost completely as hippuric acid after systemic administration.

S. cerevisiae was used as a model system for testing antioxidant or prooxidant properties of benzoic acid and other weak organic acid food preservatives (Piper, 1999). Cell sensitivity to these acids was enhanced by aerobic rather than anaerobic growth conditions. The food preservatives were shown to have a strong prooxidant effect on aerobically grown yeast cells. They were also

S. cerevisiae was used as a model system for testing antioxidant or prooxidant properties of benzoic acid and other weak organic acid food preservatives (Piper, 1999). Cell sensitivity to these acids was enhanced by aerobic rather than anaerobic growth conditions. The food preservatives were shown to have a strong prooxidant effect on aerobically grown yeast cells. They were also

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