C ITRIC A CID

Antimicrobial Properties

Inhibition of L. monocytogenes was achieved in trypticase soy yeast extract broth (TSBYE) adjusted to pH 5.0 with propionic acid; 4.5 with acetic and lactic acids; and 4.0 with citric and hydrochloric acids (Conner et al., 1990). The effect was temperature dependent in that survival of L. monocyto- genes decreased to undetectable levels within 1 to 3 weeks at 30°C, whereas at 10°C, L. mono- cytogenes survived after 11 to 12 weeks in TSBYE adjusted with acetic, citric, and propionic acids and for 6 weeks in media containing HCl or lactic acid. Similar pH-temperature relationships were noted by Cole et al. (1990), who determined that the minimum pH values that allowed survival of L. monocytogenes after 4 weeks were pH 4.66 at 30°C, 4.36 at 10°C, and 4.19 at 5°C. The minimum pH that permitted growth within 60 days (>100-fold increase in cell numbers) was the same at 30°C but increased to 4.83 at 10°C and 7.0 at 5°C. These experiments used citrate phosphate buffer in which the citric acid content was calculated at 1.1% at pH 4.4 and 0.7% at pH 6.0. The authors suggested the inhibitory activity might be the result of the chelating ability of citric acid. Additional interactive effects were also noted in the presence of salt. Sorrells et al. (1989) reported that on an equal molar basis, citric acid was more inhibitory than lactic acid, followed by acetic acid. Inter- action was demonstrated between citric acid and sodium citrate added to BHI broth adjusted to pH 4, 5, 6, and 7 (Buchanan and Golden, 1994). Rate of inactivation of L. monocytogenes after exposure to the acid combinations was dependent on pH and acid concentration whereby pH 5 and 6 appeared to be protective against inactivation, but lower pH levels were toxic.

The inhibitory capacity of hydrochloric, citric, acetic, lactic, propionic, and phosphoric acid was compared in TSB for Y. enterocolitica based on concentration of acid, pH, and degree of dissociation. A comparison of equimolar concentrations identified citric acid as the most antimicro- bial, followed by hydrochloric, lactic, phosphoric, propionic, and acetic. Based on pH, the highest inhibitory activity was associated with propionic, then lactic, acetic, citric, phosphoric, and hydro- chloric. The activity of the undissociated portion of the acid was hydrochloric followed by lactic propionic, and acetic (Brackett, 1987).

Citric acid at a concentration of 0.5% (pH 4.5) was more effective than lactic acid (pH 3.4) in inhibiting Arcobacter butzleri in a broth system (Phillips, 1999). Sodium citrate was also more effective than 2% sodium lactate, and the combination of 1.5% sodium citrate and 1.5% sodium lactate was equally inhibitory as sodium citrate alone, indicating that citrate was the more effective compound. Citric acid was more inhibitory to thermophilic bacteria than acetic or lactic acids; however, pH was not considered a reliable indicator of preservation effectiveness (Fabian and Wadsworth, 1939; Fabian and Graham, 1953). Citric acid was particularly inhibitory to flat-sour organisms isolated from tomato juice. Little bacteriostatic activity occurred at pH 5.0, but inhibition increased with decreasing pH (Murdock, 1950). When tomato juice was adjusted with citric acid to pH 4.0 and 3.7 and stored for 7 days at 5°C, lower aerobic plate counts of 3.8 and 2.6 log/ml were seen, respectively, compared to counts in unacidified juice (6.22 log/ml); aerobic counts for pH 3.7 juice incubated at 20°C were 6.12 log/ml. Yeast and mold counts were the reverse with counts of 2.09, 2.13, and 1.64 log/ml, respectively; aerobic counts at 20°C for pH 3.7 juice were 4.42 log/ml (Bizri and Wahem, 1994).

Acidification of foods before canning is often used to reduce the thermal process time of foods that are particularly sensitive to changes in sensory qualities, such as texture or appearance. Canned tomatoes acidified with citric, fumaric, or malic acids did not change in physical or chemical attributes during processing compared with unacidified tomatoes (Schoenemann and Lopez, 1973; Schoenemann et al. (1974). Okra, canned in a brine containing acetic, citric, lactic, malic, or tartaric acid to achieve an equilibrium pH of 4.3, was processed for 30 minutes in boiling water. Acidifi- cation impaired the color but enhanced the flavor of canned okra. All acids would be effective antibotulinal agents at that pH level (Nogueira et al., 1997).

Beelman et al. (1989) developed an acid-blanch-chelate (ABC) process designed to reduce mesophilic and thermophilic bacterial spoilage in canned mushrooms. They found that acid blanch- ing in a citric acid (0.05 M) solution buffered to pH 3.5 followed by canning in a solution containing 200 ppm ethylenediamine tetraacetic acid (EDTA) controlled the outgrowth of spores of C. sporo- genes PA3679 after a sublethal heating process (Okereke et al., 1990a). By adding citric acid to can brine, this process reduced thermophilic spoilage from 68% to 23.9% and spoilage was further reduced to 16.8% with the addition of EDTA. Only 2.4% of cans containing mushrooms vacuum hydrated by the ABC process spoiled (Beelman et al., 1989; Okereke et al., 1990b). This process improved quality and increased the microbial stability of canned mushrooms (Okereke and Beelman, 1990).

Mushroom extract acidified with citric acid to pH 6.7 (control), 6.22, 5.34, and 4.65 and asparagus with added citric acid or glucono-delta-lactone to pH levels of 5.9, 5.4, 5.1, 4.8, and 4.5 were inoculated with C. sporogenes PA 3679 spores. Extracts were then heated at 110°C, 115°C, 118°C, and 121°C. Although the heat resistance of C. sporogenes decreased with decreasing pH levels, heat resistance was not affected by higher processing temperatures, leading the authors to conclude that the extracts have a protective effect on C. sporogenes, thereby leading to its survival regardless of the change in pH (Ocio et al., 1994; Silla Santos et al., 1992).

Reduction in the thermal process time of canned liver paste was achieved through the addition of 0.14% citric acid (CA), 0.29% CA, 0.31% CA–2.0% trisodium citrate (TCA), or 0.31% CA–2.0% TCA-0.1% potassium sorbate (Houben and Krol, 1991). Liver pastes inoculated with 106 _{to 10}7

Bacillus spores per g and 104 C. sporogenes spores per g were heated to an F

0 of 0.05, 0.3, and

0.85. Microbial stability was achieved in liver paste formulated with 0.14% CA (pH 5.69) and processed with an F0 of 0.3. The addition of potassium sorbate did not enhance stability.

Growth curves developed for B. cereus strains grown in model vegetable broths were conducted at typical refrigeration and abuse temperatures of 5°C, 8°C, 12°C, and 16°C (Valero et al., 2000; 2003). B. cereus grew in zucchini broth (pH 6.5) at 12°C but did not grow in broth acidified to pH 5.0 at temperatures below 16°C. Growth in carrot puree was temperature dependent because B. cereus could grow at pH 5.1 and 5.2, provided temperatures were at 16°C, but could grow at 8°C only when the pH was higher (5.4 and 5.5), indicating a synergistic effect between pH and temperature. Both vegetable broths stimulated germination and growth of spores compared to nutrient broth.

Citric acid dips for foods not only retard some spoilage but also act as a chelator of metal ions responsible for enzymatic browning reactions. Cabbage and carrots were pretreated with the addi- tion of a reduced calorie dressing or with a dip of 0.2% or 1.0% citric acid for 5 or 30 minutes. The products were packed with or without modified atmosphere and stored at 4°C for 10 to 21 days. Samples pretreated with 1% citric acid displayed significantly lower total numbers, coliforms, and lactic acid bacteria counts than samples dipped with the lower concentration of citric acid. Samples containing the reduced calorie dressing were even lower in microbial numbers than acid- dipped vegetables (Eytan et al., 1992). Notermans et al. (1985) dipped potatoes in a solution of 1% citric acid and 2% ascorbic acid for 2 minutes before vacuum packing and cooking. This process inhibited C. botulinum type B when held for 70 days at 15°C or 14 days at 20°C. Citric acid dips (0.3 g/kg) for crawfish (Procambarus clarkii) stored at 4°C were not effective in retarding growth of L. monocytogenes (Dorsa et al., 1993).

Homemade mayonnaise has been a significant health hazard as a result of contamination by Salmonella from raw eggs (Membre et al., 1997; Xiong et al., 1999). The preparation of these products included adjustment of the pH with acetic acid to 3.4 to 4.1 with holding temperatures between 18°C and 20°C for up to 3 days before consumption. Mayonnaise made with >5% citric acid solution adjusted to pH below 4.05 led to more inactivation of S. Enteritidis PT4 at 22 than 5°C, but the shelf life was shortened at this higher temperature. The authors recommended that the final pH should be <3.3 when at least 20 ml of lemon juice (citric acid) per fresh egg yolk was used. If the amount of lemon juice was increased to 20 to 35 ml per egg yolk, the product should

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be held at 22°C for longer than 72 hours; when 35 ml lemon juice per egg yolk was added, the time decreased to 48 hours before use. Radford and Board (1993) found that acetic acid was more inhibitory than citric acid and recommended that mayonnaise be prepared with vinegar to a pH or 4.1 or less. The addition of garlic or mustard increased the death of S. Enteritidis, but salt had a protective effect on this organism.

C. botulinum showed no change in numbers when inoculated in tomato puree, shrimp puree, or shrimp and tomato sauce acidified with citric or acetic acids to pH 4.2 or 4.6. No toxin was detected in any of these foods over an 8-week shelf life (Post et al., 1985). Tsang et al. (1985) found no outgrowth or toxin production from spores of types A and B in TPGY medium acidified to pH 4.6 and incubated at 35°C; however, type E grew and produced toxin at 26°C in media containing citric acid at pH 4.2. When acetic acid was used, no growth occurred at pH 5.0.

Skim milk acidified with citric acid was more inhibitory to S. Typhimurium than lactic and hydrochloric acids (Subramanian and Marth, 1968). As little as 0.3% citric acid lowered the level of Salmonellae on poultry carcasses, and rinsing with a citric acid solution adjusted to pH 4.0 reduced the numbers of P. fluorescens on beef carcasses (Thomson et al., 1967); however, it did not appear to affect attachment (Appl and Marshall, 1984).

Mangoes are subject to spoilage through infection with Penicillium cyclopium (Palejwala et al., 1984). Sabouraud medium was adjusted with 0.125% to 4% citric acid or 0.016% to 1% malic acid as the sole carbon source because these acids predominate at these concentrations in a ripening mango. Healthy tissue had a pH of 5.8, whereas infected tissue was around 4.8. During ripening, the level of acidity decreased and sugars and nitrogenous compounds increased, leading to enhanced growth.

Ascospores of heat-resistant molds are difficult to kill during processing of fruit products and an increase in processing temperature can lead to adverse changes in quality (Rajashekhara et al., 1998). Survival studies for Neosartorya fischeri ascospores were conducted in mango and grape juices and heated at 75°C, 80°C, 85°C, and 90°C. Ascospores were able to withstand more than 6 hours of heating at 75°C, 5 hours at 80°C, and 3 to 4 hours at 85°C. Thermal resistance studies were conducted in mango juice adjusted to pH 3.5 with citric, lactic, malic, or tartaric acid. Citric acid provided the maximal destruction of ascospores at 85°C. Similar destruction values were seen with potassium sorbate and sodium benzoate. A level of 0.75% for both citric and lactic acids had a slight growth-retarding effect on A. parasiticus; a 0.25% concentration greatly reduced toxin production. A level of 0.5% citric or 0.75% lactic acid inhibited growth of Aspergillus versicolor, but 0.25% citric acid and 0.5% lactic acid prevented toxin production. A 0.75% concentration of either acid did not affect the growth of P. expansum (Reiss, 1976).

Sodium citrate can also exhibit bacteriostatic activity. Sodium citrate in concentrations of 0.1% to 4.0% was not as inhibitory to Streptococcus agalactiae when added to skim milk or fresh milk as citric acid at concentrations of 1%, 2%, and 4%. The MIC of 0.8% gave a pH of 4.08 to 4.12 (Sinha et al., 1968). Concentrations of 12.0% to 12.5% sodium citrate were inhibitory to Salmonella anatum and Salmonella oranienburg (Davis and Barnes, 1952).

S. aureus showed inhibition both with increasing concentrations of citric acid and decreasing pH (Minor and Marth, 1970). Citrate inhibition of S. aureus was overcome by adding calcium and magnesium ions, suggesting that citrate was a chelator of ions essential for growth (Rammell, 1962). The amount of sodium citrate had a dual effect on Lactobacillus casei. In concentrations of 12 to 18 µM/ml, sodium citrate stimulated growth, and in concentrations greater than 40 µM/ml, sodium citrate inhibited growth. Branen and Keenan (1970) also suggested that citrate chelation of metal ions inhibited L. casei. The metabolic effects of citrate on Arthrobacter species were twofold. With Arthrobacter pascens, the function of citrate appeared to be one of chelation of metal ions, even in media containing up to 1% citrate. However, with Arthrobacter simplex, citrate appeared to inhibit glucose utilization (Imai et al., 1970).

Using the multiple-barrier concept, hard-cooked eggs allowed to equilibrate in 0.5%, 0.75%, or 1.0% concentrations of citric acid and 0.2% sodium benzoate were held for 30 days at 4°C or

in 0.75% citric acid alone for 21 days at 4°C. A concentration of 0.75% citric acid was sufficient to reduce inoculated populations of S. Typhimurium, Y. enterocolitica, E. coli, and S. aureus (Fischer et al., 1985). Daly et al. (1973) used a combination of 0.1% citric acid and 0.75% glucono-delta- lactone incorporated into sausage before fermentation. This combination inhibited the growth of S. aureus early in the fermentation and the increased acidity of the meat mixture allowed the fermentation to proceed more quickly. Restaino et al. (1982) used a combination of 0.05% potassium sorbate and citric acid at pH 5.0 to reduce the growth rate of S. rouxii and Saccharomyces bisporus. Potassium sorbate at 0.2% concentration in combination with citric or lactic acid at pH 5.5 depressed growth of L. plantarum but did not affect P. aeruginosa (Restaino et al., 1981).

Toxicology

Acute toxicity data for citric acid in mice, rats, and rabbits indicated varied tolerance levels by different routes of administration (Table 4.2). Studies by Yokotani and coworkers (1971) compared toxicities of commercial citric acid and Takeda citric acid, a by-product of yeast fermentation. Death by all routes and in all animals resulted from respiratory or cardiac failure and hemorrhaging of the gastric mucosa. Gruber and Halbeisen (1948) used comparable levels of acid but suggested that many of the symptoms produced by high levels of citric acid resembled those of calcium deficiency. Horn et al. (1957) evaluated dosage levels in mice given the acid intravenously. Deaths resulted from acute acidosis and lung hemorrhages. Short-term studies on rats fed 0.2%, 2.4%, and 4.8% citric acid in a commercial diet showed lowered weight gains as a result of decreased intake of food and minor blood chemistry abnormalities at the 4.8% level; slight atrophy occurred in regions of the thymus and spleen (Yokotani et al., 1971).

Long-term studies involving rats fed quantities up to 1.2%, 3%, or 5% citric acid (Bonting and Jansen, 1956; Horn et al., 1957) showed no abnormalities other than lowered weight gains and feed consumption in the 5% group. Cramer et al. (1956) fed vitamin D-free diets containing low calcium levels but adequate levels of phosphorus supplemented with 0.02 mol sodium citrate and citric acid. Although there was no effect on weight gain, the urine contained calcium citrate. The authors concluded that citrate had a rachitogenic effect on the test animals. Dalderup (1960) fed rats 1.5, 4.5, or 12 g citric acid per kg of a noncariogenic diet to show the possible effect of citric acid on the development of teeth. The number of cavities formed did not differ between the control and experimental groups; however, the highest dosage contributed to enamel erosion.

Acute toxicity data of sodium citrate are shown in Table 4.2. Subcutaneous injections of 320 to 1200 mg/kg body weight of sodium citrate into dogs decreased blood calcium levels while increasing calcium levels in urine (Gomori and Gulyas, 1944). In another study, 7.7% sodium citrate (5% citric acid) fed to rabbits for 60 days produced no unusual effects (Packman et al., 1963).

Application and Regulatory Status

Citric acid is a tricarboxylic acid having a pleasant sour taste. It is highly water soluble and enhances the flavor of citrus-based foods. It is approved for use in ice cream, sherbets and ices, beverages, salad dressings, fruit preserves, and jams and jellies, and it is used as an acidulant in canned vegetables and dairy products. It is a precursor of diacetyl and therefore indirectly improves the flavor and aroma of a variety of cultured dairy products. It can control the pH for optimum gel formation. Citric acid also acts synergistically with antioxidants to prevent rancidity by chelating metal ions (Gardner, 1972). Citric acid is approved as a GRAS substance for miscellaneous and general-purpose usage when used in accordance with good manufacturing practices, in the acid form (21 CFR 184.1033) or as the calcium (21 CFR 184.1195), potassium (21 CFR 184.1625), or sodium salt (21 CFR 184.1751). The acceptable daily intake for humans is listed in Table 4.3.

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