P ROPIONIC A CID

Antimicrobial Properties

Propionic acid inhibited spore-forming bacteria, especially rope bacteria (B. subtilis), at pH 6.0. As the pH decreased to 5.0 or 4.0, the acid inhibited yeasts and molds but not to the same extent as bacteria (Woolford, 1975b). Chung and Goepfert (1970) compared various organic acids to determine the highest pH level that inhibited growth of S. anatum, Salmonella Senftenberg, and

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Salmonella Tennessee when all other parameters were at their optimum. Propionic acid at pH 5.5 inhibited the growth of Salmonellae at a higher pH level than other acids (pH 5.4, acetic; pH 5.1, adipic; pH 4.6, succinic; pH 4.4, lactic; pH 4.3, fumaric and malic; pH 4.1, tartaric; pH 4.05, citric). Cherrington et al. (1991) determined that 0.5 to 0.7 M propionic acid at pH 5.0 was more inhibitory in 60 minutes compared to formic acid in 3 hours for E. coli K12 and Salmonella. At a 5-mM concentration, the rate of RNA (ribonucleic acid), DNA (deoxyribonucleic acid), protein, lipid, and cell-wall synthesis decreased, which led to an increase in cell mass without cell division and a decrease in viability (Cherrington et al., 1990). Eklund (1985) concluded that propionic acid was not as inhibitory as benzoic acid for P. aeruginosa. Much higher concentrations of propionic acid were bacteriostatic to B. subtilis, E. coli, S. aureus, and Candida albicans.

The salts of propionic acid are also effective antimicrobial agents. Calcium propionate prevented rope formation caused by B. mesentericus (subtilis) in bread dough at a level of 0.188%, pH 5.8, or at a level of 0.156%, pH 5.6 (O’Leary and Kralovec, 1941). Incorporation of calcium propionate into coatings for ready-to-eat products was effective in reducing problems with L. monocytogenes (Janes et al., 2002). Zein (Z) film coatings were dissolved in propylene glycol (ZP) or ethanol (ZE) with and without nisin (N; 1,000 IU/g) or calcium propionate (CP; 1%) and coated onto ready-to- eat chicken samples dipped in L. monocytogenes. Products were stored for up to 24 days at 4°C or 8°C. Nondetectable levels of the pathogen were found in products ZEN, ZPNCP, or ZENCP.

Sodium propionate at levels of 0.1% to 5% delayed growth of S. aureus, Sarcina lutea, Proteus vulgaris, L. plantarum, Torula species, and S. ellipsoideus by as much as 5 days. This substance not only postponed spoilage in fresh figs, syrup, applesauce, cherries, and berries but also prevented spoilage in vegetables having a more neutral pH, such as lima beans and peas (Wolford and Anderson, 1945).

Turkey breast meat was formulated with sodium salts of lactate, acetate, pyruvate, citrate, and propionate to a target level of pH 6.0, inoculated with C. botulinum, and held at 28°C for 0 to 18 days. At periodic intervals, samples were removed from storage and tested for production of neurotoxin. Toxin production occurred after 2 days for pyruvate, 3 for citrate, 4 for lactate and acetate, and 5 with a 2% concentration. At a 6% concentration, toxicity occurred after 7 days for pyruvate; 18 days for citrate; and greater than 18 days for propionate, acetate, and lactate. Citrate was most effective based on molarity (Miller et al., 1993).

In laboratory studies examining the effect of sodium propionate on L. monocytogenes, the organism was able to grow at up to a 0.3% concentration in tryptose broth adjusted to pH 5.6 and incubated at 4°C, 13°C, 21°C, and 35°C. Reduction of pH to 5.0 minimized growth at 13°C, 21°C, and 35°C and prevented growth at 4°C (El-Shenawy and Marth, 1989a). When media containing 0.3% sodium propionate was adjusted to pH 5.6 with acetic, tartaric, citric, or lactic acids in media (El-Shenawy and Marth, 1992), the lag phase of L. monocytogenes grown at 13°C was extended by 7, 7, 5, and 4 days, respectively. At pH 5.0 and 13°C, the levels of L. monocytogenes were undetectable. Sodium propionate at a 0.3% concentration was also effective at pH 5.0 in reducing numbers of L. monocytogenes when used in conjunction with acetic acid in cold-pack cheese food (Ryser and Marth, 1988). Exposure of L. monocytogenes to 8% sodium propionate for 60 minutes caused injury (Buazzi and Marth, 1992).

Emphasizing the multiple-barrier concept in retarding growth, Golden et al. (1995) examined the combination of sodium propionate and SA at a number of concentrations, pH levels, and temperatures. A three-strain mixture of L. monocytogenes was inoculated into BHI broth containing 0.08% EDTA, 0.02% ascorbic acid, and 0.9% sodium propionate or SA; adjusted to pH 4.5, 4.0, 3.5, or 3.0 with hydrochloric acid; and stored at 28°C, 19°C, or 4°C. Inactivation of L. monocyto- genes was the result of incubation temperature, concentration of the organic acid, degree of dissociation of the acid, and pH. Propionic acid was more effective than acetic acid as an anti- microbial, particularly at the higher pH level. The acids contributed to approximately 85% of the antimicrobial activity with 14% contributed by ascorbic acid; little activity was ascribed to EDTA in combination with the acids.

The effect of fat concentration on the efficacy of antimicrobial agents against L. monocytogenes was studied in pork liver beaker sausage (Hu and Shelef, 1996). Sausage batter was mixed with 0.2% sodium propionate, 1.8% sodium lactate, and 0.1% sorbic acid or potassium sorbate. Increas- ing the fat content from 22% to 67% decreased the growth of L. monocytogenes by only 1.5 logs. The antimicrobial activity of sodium propionate was most affected at 4°C by fat concentration with a 1.5 log reduction of L. monocytogenes at 22% fat compared to 2.8 log reduction at 67% fat; with sodium lactate, 0.5 and 1.8 log reductions were observed, respectively.

Small amounts of β-alanine could overcome bacteriostatic action of sodium propionate for E. coli. However, β-alanine could not reverse the inhibitory effect of propionic acid in Aspergillus clavatus, B. subtilis, Pseudomonas species, or Trichophyton mentagrophytes. The inhibitory action could be the result of an interference with β-alanine synthesis (Wright and Skeggs, 1946).

Propionic acids and their salts are primarily inhibitory to molds; however, some species of Penicillium grew on media containing 5% propionic acid (Heseltine, 1952b). Propionic acid at a concentration of 0.1%, pH 4.5, reduced growth and aflatoxin formation of A. flavus; at 0.2%, no growth occurred. This inhibitory effect was more pronounced with the addition of acid at the time of inoculation, rather than later (Ghosh and Häggblom, 1985).

Propionic acid at a concentration of 2435 ppm was more effective than acetic acid in limiting growth of Fusarium oxysporum but less effective than sorbic acid and potassium sorbate. Spore germination was inhibited by 1402 ppm (Tzatzarakis et al., 2000).

Concentrations of propionic acid and propionates ranging from 8% to 12% were effective in controlling mold growth on the surface of cheese and butter (Deane and Downs, 1951; Ingle, 1940). A 5% calcium propionate solution acidified to pH 5.5 with lactic acid was as effective as a 10% unacidified solution in preventing surface mold growth on butter (Olson and Macy, 1945). Molds were inhibited by less calcium propionate by weight than sodium propionate. Not only was the final pH of the substrate critical, but also various organisms displayed different tolerances to the compounds (Olson and Macy, 1940).

A novel use of propionic acid to retard spoilage of bread was investigated by Gardner et al. (2001). Adding yeast extracts previously fermented by Propionibacterium freudenreichii provided a source of propionic acid in bread formulation. Bread produced with the extracts contained less ethanol and supported less spoilage by molds.

Mold spoilage is a significant economic problem in bakery products. Although mold spores are destroyed during baking, postprocess contamination from the atmosphere, cooling surfaces, and wrapping materials reintroduce molds. Eurotium amstelodami, herbariorum, rubrum, and repens were inoculated by needle into cake analogues prepared with calcium propionate, sodium benzoate, and potassium sorbate at a level of 0.3% by weight. All preservatives were effective at pH 6.0 and a water activity of 0.8 to 0.85 (Guynot et al., 2002). Subsequent studies (Marin et al., 2002a,b) examined several levels of calcium propionate, potassium sorbate, and sodium benzoate (0.003%, 0.03%, and 0.3%) at pH 4.5, 6.0, and 7.5 and water activity of 0.8, 0.85, and 0.93 in a model agar system on the retardation of growth of E. amstelodami, herbariorum, and rubrum, A. flavus and niger, and Penicillium corylophilum. None of the preservatives were effective at neutral pH. Suboptimal doses (0.03%) led to enhanced growth of Aspergillus and Penicillium isolates.

Toxicology

Orö and Wretland (1961) determined the LD50 of propionic acid for mice, and Hara et al. (1963)

determined the LD50 of calcium and sodium propionate for rats (Table 4.2). Studies involving albino

rats fed propionic acid at 50 cm3_{/kg of rice for 110 days showed umbilicate or warty lesions on}

the stomach (Mori, 1953). Additional studies using calcium and sodium propionate fed to mice, rats, and humans showed no toxic effects (Graham et al., 1954; Graham and Grice, 1955; Hara, 1965; Harshbarger, 1942). There is evidence that the sodium salt has some local antihistaminic activity (Heseltine, 1952a).

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Application and Regulatory Status

Propionic acid is a monocarboxylic acid with a slightly pungent, disagreeable odor. Salts of the acid have a slight cheeselike flavor. The acid form is readily miscible with water, and the sodium salt is more soluble than the calcium form. It is normally found in cheese and as a metabolite in the ruminant gastrointestinal tract. Swiss cheese contains up to 1% propionic acid because of the growth and metabolism of propionibacteria, which are associated with its manufacture and the characteristic Swiss cheese flavor. This naturally formed additive becomes a developed preservative that limits mold growth on Swiss cheese. This additive is also used as a mold inhibitor in cheese foods and spreads. Its antimicrobial effect is limited to most yeast and bacteria.

Propionic acid and its salts are used primarily as mold and rope inhibitors in bread. Baking destroys most molds, but surface recontamination can occur during packaging, and growth can be seen under the wrapper during storage. Propionates can be added to bread dough without interfering with leavening because there is little or no effect on yeast. Sodium propionate is recommended for use in chemically leavened products because the calcium ion interferes with the leavening action. Calcium propionate is preferred, however, for use in bread and rolls because the calcium contributes to the enrichment of the product (Chichester and Tanner, 1972).

Propionic acid (21 CFR 184.1081) and its salts, calcium (21 CFR 184.1221) and sodium propionate (21 CFR 184.1784), are approved as GRAS substances for miscellaneous and general- purpose usage. In addition, calcium and sodium propionate are listed as antimycotics when migrat- ing from food-packaging material (21 CFR 181.23). No upper limits are prescribed for use of this additive, except bread and rolls, which conform to standards of identity. A limit of 0.32% can be used in flour and in white bread and rolls, 0.38% in whole-wheat products, and 0.3% in cheese products (Robach, 1980). The acceptable daily intake for humans is listed in Table 4.3.