364

Journal of Food Protection, Vol. 48, No. 4, Pages 364-375 (April 1985) Copyright5 International Association of Milk, Food, and Environmental Sanitarians

Growth and Inhibition of Microorganisms in the Presence of Sorbic Acid: A Review

MICHAEL B. LIE WEN and ELMER H. MARTH*

Department of Food Science and The Food Research Institute, University of Wisconsin-Madison, Madison, Wisconsin 53706 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/48/4/364/1657271/0362-028x-48_4_364.pdf by guest on 29 September 2021 (Received for publication July 9, 1984)

ABSTRACT food product to which preservatives may be added. No Sorbate (sorbic acid) generally is an effective inhibitor of upper limits are imposed for foods not covered by Fed­ most molds and yeasts and some bacteria. Environmental fac­ eral Standards of Identity. Thus, in natural cheese, the tors such as pH, water activity, temperature, atmosphere, maximum quantity may not exceed 0.3% by weight, cal­ microbial load, microbial flora and certain food components can culated as sorbic acid, and the maximum is set at 0.2% influence the effectiveness of sorbate. Strains of microor­ in pasteurized blended cheese, pasteurized cheese spread, ganisms resistant to sorbate exist and therefore are common cold pack cheese and cheese food and spread (39). causes of food spoilage. Some molds and bacteria are able to degrade sorbate. This paper reviews the factors that affect the antimicrobial effectiveness of sorbate in foods. APPLICATIONS

Sorbic acid is a straight chain a, p-unsaturated monocarboxylic acid and has the structure

Sorbic acid was first isolated from the pressed un- CH3CH = CHCH = CHCOOH (179). The carboxyl group ripened berry of the rowan or mountain ash tree in 1859 reacts readily and forms salts and esters. The salts are by the German chemist A. W. Hoffmann (6). It was not important in food applications because of their high solu­ until 1939, however, that E. Miiller and C. M. Gooding, bility in water. Potassium sorbate was specifically de­ working independently in Germany and the United States, veloped to prepare the aqueous stock solutions needed for respectively, discovered the antimicrobial activity of sor­ dip, spray and metering applications (36, 65). Sorbic acid bic acid (95). In 1945, Gooding was awarded a U.S. pa­ is only slightly soluble in water; however, it is more sol­ tent for use of the antifungal properties of sorbic acid uble in fats than is potassium sorbate. Sorbic acid can (64), and since the mid-1950s it has been used to a grow­ be incorporated with dry materials by mixing with salt, ing extent throughout the world to protect a variety of flour or corn starch. It can be solubilized with sodium foods against deterioration by microorganisms. or potassium hydroxide, or it may be dissolved in prop­ ylene glycol or ethanol for use in dips or sprays (36). REGULATORY STATUS Since sorbic acid sublimes upon heating, it should be added to foods only after any prolonged boiling or heat­ Because sorbic acid was not used as a food preserva­ ing that may be employed in food processing (6, 36). tive until a time when extensive toxicological investiga­ Sorbic acid and its potassium salt are the most widely tions were required for new food additives, it is among used forms of the compound and are collectively known the most thoroughly investigated of all preservatives. It as sorbates. Their most common use is preservation of was found harmless in numerous acute, subchronic and food, animal feed, cosmetic and pharmaceutical products chronic toxicity tests (52, 61, 94). Sorbic acid is as well as technical preparations that come in contact metabolized in the body like other fatty acids. In humans with food or the human body. Methods of application in­ and animals, it is subject to B-oxidation typical of fatty clude: direct addition into the product; dipping, spraying, acid degradation. The half-life in the body is 40-110 min, or dusting of the product; or incorporation into the wrap­ depending on the dosage, and under normal conditions per (6, 7, 158). it is completely oxidized to C02 and H20 (53, 95). Typical use levels in foods range from 0.02% in wine Use of sorbic acid in foods is permitted in most coun­ and dried fruits to 0.3% in some cheeses (Table 1). tries which regulate their food supply (95). The Foods in which sorbate has commercially useful antimic­ maximum permissible level, other than in exceptional robial activity include baked goods (cakes and cake situations, is between 0.1 and 0.2%. In the U.S., sorbic mixes, pies and pie fillings, doughnuts, baking mixes, acid is a GRAS substance and its use is permitted in any fudges, icings), dairy products (natural and processed

JOURNAL OF FOOD PROTECTION, VOL. 48, APRIL 1985 SORBIC ACID INHIBITS MICROBIAL GROWTH 365

TABLE 1. Typical concentration (%) of sorbic acid used in proaches its dissociation constant (pKa), which is 4.75. various food products. At this pH value, 50% of sorbic acid is in the effective Cheeses 0.2-0.30 undissociated form (161) (Table 2). Therefore, sorbate is Beverages 0.03-0.10 more effective in foods with low rather than high pH Cakes and pies 0.05-0.10 values (6, 7, 11, 14, 95, 161). The upper pH limit for Dried fruits 0.02-0.05 activity of sorbate is 6.0-6.5, while those for propionate Margarine (unsalted) 0.05-0.10 and benzoate are 5.0-5.5 and 4.0-4.5, respectively. Many Mayonnaise 0.10 investigators have demonstrated the importance of using Fermented vegetables 0.05-0.20 sorbate at proper pH values. For example, Park and Jams and jellies 0.05 Marth (119) showed that Salmonella typhimurium would Fish 0.03-0.15 grow in nutrient broth (pH 6.7) or skimmilk (pH 6.4) Semi-moist pet food 0.1-0.3 fortified with 0.3% sorbic acid when the media were not Wine 0.02-0.04 acidified. However, when the pH was reduced to 5.0, Fruit juices 0.05-0.20 growth did not occur in either medium and, in time, a Downloaded from http://meridian.allenpress.com/jfp/article-pdf/48/4/364/1657271/0362-028x-48_4_364.pdf by guest on 29 September 2021 major portion of the population was inactivated. cheeses, cottage cheese, sour cream, yogurt), fruit and berry products (artificially sweetened confections, dried TABLE 2. Effect of pH on sorbic acid dissociation" fruits, fruit drinks, jams, jellies, wine), vegetable prod­ pH Undissociated (%) ucts (olives, pickles, relishes, salads) and other miscel­ 7.00 0.6 laneous food products (certain fish and meat products, 6.00 6.0 mayonnaise, margarine, salad dressings) (6, 108). 5.80 7.0 5.00 37.0 4.75 (pKa) 50.0 ENVIRONMENTAL FACTORS AFFECTING EFFEC­ 4.40 70.0 TIVENESS OF SORBATE 4.00 86.0 3.70 93.0 Environmental factors such as pH, water activity (aw), 3.00 98.0 temperature, atmosphere, initial microbial load, type of "From Sofos and Busta (148). microbial flora, and certain food components, singly or in combination, can influence the antimicrobial activity of sorbate. Together with preservatives such as sorbic acid, they often act to broaden antimicrobial action or Water activity increase it synergistically. Use of other preservatives in Addition of substances to food that reduce its aw have combination with sorbate can also broaden or intensify a beneficial effect on the action of antimicrobial preserva­ antimicrobial action. The length and temperature of stor­ tives (10). The most important substances to consider are age are other important considerations. If growth of spoil­ salt and sugar (87). Both intensify the action of sorbate age or pathogenic microorganisms is inhibited, but the primarily by reducing aw. They may also induce cellular microorganisms are not killed, growth will eventually re­ swelling, which makes many microorganisms more sus­ sume under proper conditions. The length of inhibition ceptible to action of preservatives (95). Costilow et al. will vary with storage temperature as well as with any (42-44) noted that salt (NaCl) increased the effectiveness of the other factors discussed. of sorbate in inhibiting spoilage yeasts in cucumber fer­ mentations. Sheneman and Costilow (155) saw the same Effects ofpH effect with sucrose in sweet cucumber pickles. Robach Organic acids such as sorbic acid dissociate in aqueous and Stateler (148) found that certain combinations of solutions and release hydrogen ions. Some food preserva­ NaCl and sorbate resulted in a synergistic inhibition of tives such as acetic acid act mainly by lowering the pH Staphylococcus aureus. Acott et al. (1) showed that suc­ of the environment to the point where microorganisms rose (aw =0.85) and sorbate in combination inhibited can no longer grow. Although the undissociated form of growth of Aspergillus glaucus and Aspergillus niger and acetic acid has antimicrobial action, it is primarily the slowed the growth of Staphylococcus epidermidis. At an hydrogen ion and the resulting decrease in pH that in­ aw of 0.88, all three organisms were able to grow, but hibits microorganisms (45, 91). at a slower rate than in the absence of sorbate. Gooding With sorbic acid and other organic acids, it is the un­ et al. (65) indicated that salt and glucose have a marked dissociated molecule that provides antimicrobial activity synergistic effect on fungistasis by sorbic acid. In high (45, 95, 99). The amount of the molecule in the undis­ sugar systems, increased antimicrobial activity occurred sociated form is determined by pH. This, along with sol­ even at pH values above 6.5. In contrast, Beuchat (16) ubility properties, determines the foods in which organic reported that the presence of sucrose or NaCl can, in acids may effectively be used (135). The antimicrobial some instances, reduce the synergistic effect of heat and effectiveness of sorbate increases as the pH value ap- sorbate on inactivation of molds.

JOURNAL OF FOOD PROTECTION, VOL. 48, APRIL 1985 366 LIEWEN AND MARTH

Temperature tion. Therefore, in fat/water emulsions a relatively high Sorbate in combination with low temperature increased proportion of sorbate remains in the microbiologically the storage life of grape juice (121). The combination of susceptible water phase. This property makes sorbate use­ sorbate and a mild heat treatment greatly extended the ful for preservation of margarine, mayonnaise and salad shelf-life of fruit products (20, 150). Beuchat (16-19, 21) dressings. However, an increase in fat content of a prod­ has shown that sorbate synergistically enhances cellular uct will lower the amount of sorbate in the aqueous phase injury during heating of several yeast and mold strains. where it is needed for microbial control (116). Soluble Shibasaki and Tsuchido (156) found that sorbate in­ food components such as salts and sugars will also de­ creased sensitivity of A. niger to heat but had no effect crease the amount of sorbate in the aqueous phase (65). on Penicillium thomii. Beuchat (16, 17) demonstrated This effect may be offset, however, by the synergistic that NaCl and sucrose can impart greater resistance to action of sorbate and salt or sugar to give an increased heat, probably because cells are dehydrated, but regard­ antimicrobial effect (1, 16, 42-44, 65, 148, 155). Some less of solute concentration sorbate treatment increased acids decrease the solubility of sorbate in water (65), but

the sensitivity of microorganisms to heat. Sorbate also re­ acids also enhance antimicrobial activity by increasing the Downloaded from http://meridian.allenpress.com/jfp/article-pdf/48/4/364/1657271/0362-028x-48_4_364.pdf by guest on 29 September 2021 tards or prevents repair of thermal injury in bacteria and proportion of sorbate in the active undissociated form. yeasts (19, 156). Tsuchido et al. (175) found that sorbate Citric and lactic acids potentiate the antimicrobial action did not inhibit recovery of nucleic acid synthesis in ther­ of sorbate to a greater extent than do inorganic acids mally injured cells of Candida utilis, but it did inhibit (140, 141, 159). Acetic acid, which is often used in con­ recovery of protein synthesis and respiratory activity. junction with sorbate in mayonnaise and salad dressings, Generally, temperatures outside the optimal growth range decreases the fat-to-water ratio of sorbate partition (65). for the microorganism in question will increase the effec­ Food ingredients that have antimicrobial activity by them­ tiveness of sorbate in inhibiting growth. selves, such as spices, can increase the spectrum or inten­ sity of activity of sorbate. Atmosphere Several investigators have shown that C02 and sorbate Preservative interactions in combination will effectively inhibit microorganisms. Mixtures of preservatives may reinforce or reduce their Elliott et al. (55, 56) showed that C02 and sorbate acting separate growth-inhibitory activities. They may work synergistically inhibited Salmonella enteriditis and S. au­ synergistically, additively or antagonistically with one reus. Carbon dioxide and sorbate will inhibit mold spoil­ another, as illustrated in Fig. 1. As an example, if two age of grains (48). Sorbate will delay the growth of compounds are combined in this preservative system and psychrotrophic bacteria in vacuum-packaged pork loins they react synergistically, only 40% of the minimum in­ (111). hibitory concentration (MIC) of each compound is re­ Microbial flora quired to give 100% inhibition of the test microorganism. The numbers and variety of microorganisms present in Conversely, in an antagonistic relationship, 50% of the a food to be preserved will affect the ability of sorbate MIC of one compound and 85% of the MIC of the other to prevent microbial growth and spoilage. Experiments compound are required to achieve 100% inhibition of have shown that the lower the initial microbial load, the growth. The type of relationship between two preserva­ longer sorbate will afford protection by preventing growth tives can vary with the proportion of each used, and with of the microbes (6, 65). A low initial microbial count allows microorganisms to be inhibited in the initial lag phase of growth and not in the exponential (log) phase where to do so requires a greater concentration of preser­ vative (95). While sorbate is effective against most yeasts and molds and some bacteria, it is not effective against all microorganisms. Some may grow in the presence of high concentrations (0.3%) of sorbate, and a few actually metabolize it. In foods with a mixed flora, some microor­ ganisms may be inhibited by sorbate, but those unaf­ fected may actually grow faster and to higher concentra­ tions when the chemical is present rather than absent be­ cause competition by some organisms has been elimi­ nated.

Food components Effectiveness of sorbate is influenced by food compo­ Figure 1. Minimum inhibitory concentration (MIC) when two nents which can affect distribution of sorbate within the preservatives are combined at various concentrations. microenvironment. In comparison with other preserva­ ADD = additive, ANT = antagonistic and SYN = synergistic. tives, sorbate has a favorable fat-to-water ratio of parti­ Adapted from Rehm (136).

JOURNAL OF FOOD PROTECTION, VOL. 48, APRIL 1985 SORBIC ACID INHIBITS MICROBIAL GROWTH 367 the microorganism to be inhibited. For example, combi­ presence of 0.3% potassium sorbate in a solid laboratory nations of benzoic acid and boric acid react antagonisti­ growth medium. cally against Escherichia coli but are synergistic in in­ In a study of moldy Cheddar cheese, Bullerman and hibiting Aspergillus spp. (10, 136). A combination of Olivigni (34) found that 82% of the isolates were of the sorbic acid and formic acid reacts antagonistically against genus Penicillium, 7% Aspergillus and 1% Fusarium. Saccharomyces cerevisiae, but is synergistic in inhibiting Seven percent of all molds were able to produce mycoto- A. niger (95, 138). Sorbic acid in combination with sul­ xins. In another study of Swiss cheese, 87% of the iso­ fur dioxide, formic acid, benzoic acid or p-hydroxyben- lates were Penicillium species and less than 1% were As­ zoic acid acts additively in inhibiting E. coli (137). A pergillus species. A total of 5.5% were able to produce mixture of sorbic acid and sulfur dioxide will act syner- mycotoxins (28). Toxic Penicillium and Fusarium species gistically to inhibit A. niger (138). The combination of are generally more capable of growth at low temperatures sorbic acid and benzoic acid has an additive effect in in­ than are toxic species of aspergilli (114). Mold growth hibiting sensitive microorganisms (10, 95, 137, 138). An on cheese is a problem during ripening as well as refrig­ additive effect does not enable a reduction in the total

erated storage (28, 29, 31, 171, 172). Downloaded from http://meridian.allenpress.com/jfp/article-pdf/48/4/364/1657271/0362-028x-48_4_364.pdf by guest on 29 September 2021 amount of chemicals used, but often the spectrum of anti­ In 1966, Marth et al. (101) demonstrated that some microbial activity is increased. molds in the genus Penicillium could grow in the pres­ The antimicrobial effectiveness of antioxidants in com­ ence of up to 0.71% (7100 ppm) potassium sorbate bination with sorbate has been investigated. Several re­ (Table 3). The molds were isolated from natural and pro­ searchers have shown that butylated hydroxyanisole cessed Cheddar cheese previously treated with sorbate. (BHA) or tertiary butylhydroquinone (TBHQ) inhibit S. Some of the penicillia degraded sorbate and produced 1, aureus and S. typhimurium more effectively when com­ 3-pentadiene, a volatile compound with an extremely bined with sorbate in trypticase soy broth (49, 84, 88, strong kerosene-like odor. Later Finol et al. (59) reported 148). Morad et al. (109) demonstrated that BHA in com­ that penicillia, also isolated from cheese, could grow in bination with sorbate acted synergistically to inhibit S. the presence of up to 1.2% (12,000 ppm) potassium sor­ typhimurium and the natural flora in cooked turkey. bate (Table 4). Lie wen and Marth (93) examined several Ahmad and Branen (2) found that BHA combined with strains of penicillia and aspergilli for their abilities to sorbate totally inhibited growth of Aspergillus flavus in grow in the presence of sorbate. They found that only a liquid growth medium. molds isolated from sorbate-treated cheeses were able to Many studies have shown that sorbate and nitrite act grow in the presence of 0.3% (3000 ppm) or more sor­ synergistically to retard botulinal toxin production and ex­ bate and none of the aspergilli they tested were able to tend the time necessary for toxin to be produced under grow in the presence of more than 0.1% (1000 ppm) sor­ abuse conditions (78, 100, 112, 113, 125, 154, 160, bate (Tables 5, 6). The aspergilli generally are more sen­ 161). For this reason, sorbate has been suggested as a sitive to sorbate than are the penicillia. partial replacement for nitrite in cured meat products. It has been postulated that nitrite and sorbate react and form TABLE 3. Amount of sorbate that permitted growth of penicil­ compounds that are more inhibitory than the parent sub­ lia isolated from sorbate-treated cheese." stances. However, these compounds have been found only in non-food systems at very low pH values and with Species of Penicillium Maximum amount of sorbate permitting very high nitrite and sorbate concentrations (112, 161). growth (ppm) Amano et al. (4) found a tylosin-sorbate combination roqueforti 5,400 effective in preserving fish sausage held at elevated tem­ roqueforti (variant) 7,100 peratures (30 and 37°C). Boyd and Tarr (26) saw that notatum 2,300 the combination of smoke and sorbate inhibited mold frequentans 2,800 spoilage in fish. Perry et al. (123) reported that the shelf- cyano-fulvum 1,800 life of eviscerated cut-up poultry was extended by the synergistic effect of acidic hydration followed by sorbate aFrom: Marth et al. (101). application. TABLE 4. Amount of sorbate that permitted growth of penicil­ lia isolated from sorbate-treated cheese". EFFECTS OF SORBATE ON GROWTH OF MOLDS Species of Penicillium Maximum amount of sorbate permitting One of the most common uses of sorbate is as an in­ growth (ppm) hibitor of mold growth on cheese and cheese products roqueforti 10,000 (6, 36), and its effectiveness has been demonstrated by cyclopium 3,000 several workers (52, 53, 102-106, 157, 158). The permit­ cyclopium (variant) 12,000 ted use level for cheeses under the Federal Standard of viridicatum 6,000 Identity is 0.2-0.3% (39). This concentration will inhibit puberulum 12,000 growth of most molds that contaminate cheeses. How­ crustosum 7,000 ever, Bullerman (29) found that about 70% of the molds lanoso-viride 3,000 isolated from moldy cheese were able to grow in the "From: Finol et al. (59).

JOURNAL OF FOOD PROTECTION. VOL. 48, APRIL 1985 368 LIEWEN AND MARTH

TABLE 5. Maximum concentration of potassium sorbate (ppmj The shelf-life of cottage cheese can be extended by which permitted growth of various penicillia at 25 and 4°Ce adding sorbate (5, 7, 40). Bradley et al. (27) found that Culture Incubation temperature 0.1% potassium sorbate inhibited growth of Penicillium 25°C 4°C frequentans, as well as other spoilage microorganisms. Other workers have also noted that sorbate inhibits spoil­ P. cyclopium-8* 3000 2000 age organisms in cottage cheese (41, 72, 122). Mold P. roqueforti-2T 9000 3000 growth was also inhibited in unsalted butter as a result P. cyclopium (atypical)-25a 9000 9000 P. puberulum-33* 9000 9000 of sorbate treatment (80, 81, 117). Mold growth in grains can be inhibited by sorbate P. viridicatum-34R 9000 2000 P. puberulum-37R 9000 6000 treatment (128, 131, 164). Danzinger et al. (48) found P. cyclopium (atypical)-40a 9000 9000 that 0.05% potassium sorbate increased the time required 5 P. crustosum-42R 6000 2000 to obtain yeast and mold counts of 1.3 x 10 /g of dry P. lanoso-viride-44a 3000 2000 weight (termed Safe Storage Time, or SST) of wet corn

P. roqueforti-KV* 9000 9000 slightly over the control. The SST was increased by al­ Downloaded from http://meridian.allenpress.com/jfp/article-pdf/48/4/364/1657271/0362-028x-48_4_364.pdf by guest on 29 September 2021 P. camemberti-%11 500 NGd most 2 d with 0.10% potassium sorbate, while 0.20% P. chrysogenum 500 500 increased the SST by 3 d. The primary organisms inhi­ P. digitalum 1000 500 bited were yeasts and molds. Sauer and Burroughs (151) P. frequentans 500 500 reported that with 0.5 and 1.0% potassium sorbate, the P. notatum 500 500 mold-free storage time for corn containing 18% moisture P. expansum 1000 NG P. roqueforti-849 2000 2000 was 2 and 3 weeks, respectively, compared to less than P. patulum-M59 1000 NG 1 week for the control. At the same levels of sorbic acid, P. sp.-K2a'c 6000 6000 the mold-free storage time was increased to 7 and 12 P. sp.-Sla-c 9000 6000 weeks, respectively. P. sp.-S2a'c 9000 9000 Sorbate also inhibits mold growth in hams (9,82,83) P. sp.-S3b-c 2000 1000 and sausage. Wallhauser and Luck (177) reported that b c P. sp.-Bl - 1000 500 0.02 to 0.04% potassium sorbate inhibited growth of As­ b c P. sp.-B2 - 1000 1000 pergillus versicolor in culture media and fermented saus­ b c P. sp.-B7 - 1000 1000 age at pH 5.7-5.9. "Isolated from sorbate-treated cheese. Bandelin (11) determined the MIC of sorbic acid re­ bIsolated from cheese not treated with sorbate. quired to inhibit several mold species. At pH 5.0, the c Mold of genus Penicillium, species not identified. MICs were 0.02% for Alternaria solani, 0.08% for dNG = no growth. e Penicillium citrinum and 0.08% for A. niger. Bullerman From Liewen and Marth (93). (31) found that potassium sorbate at 0.05, 0.10 and 0.15% delayed or prevented spore germination and initia­ tion of growth, and slowed mycelial growth by strains of Aspergillus parasiticus and A. flavus. Similar results TABLE 6. Maximum concentration of potassium sorbate (ppm) b were obtained with Penicillium patulum and P. roqueforti which permitted growth of various aspergilli at 25 and 4°C (33). Liewen and Marth (92) noted that sorbate had detri­ Culture Incubation temperature mental effects on viability of conidia of P. roqueforti. 25°C 4°C Viability decreased as the sorbate concentration increased A.flavus-93070 1000 NGa (Fig. 2). Przbylski and Bullerman (130) noted a similar A. flavus-93080 500 NG trend for the conidia of A. parasiticus. Lukas (96) re­ A.flavus-%91\1 500 500 ported that 0.1% potassium sorbate hindered germination A.flavus-W\951 500 NG of conidia of A. niger and 0.15% inhibited mycelial A.flavus-3494 500 NG growth. A. flavus-3251 500 NG A small number of molds found growing on foods pro­ A. flavus3161 500 NG duce mycotoxins (30, 107, 153). Tong (170) demonstrat­ A. flavus-WB500 500 500 ed that potassium sorbate inhibited growth and ochratoxin A.flavus-4098 500 NG A production by Aspergillus sulphureus and Penicillium A. flavus-4018 500 500 viridicatum. Bullerman (31) found that potassium sorbate A. flavusA02135 500 500 greatly reduced or prevented production of aflatoxin B] A. parasiticus-3000 500 NG A. parasiticus-]5951 500 NG by A. parasiticus and A. flavus. The same investigator A. parasiticus-3315 500 NG (32, 33) showed that potassium sorbate had a similar ef­ A. parasiticus-2999 500 NG fect on patulin production by P. patulum. However, A. niger 500 NG while potassium sorbate reduced growth by P. roqueforti, A. nidulans 500 NG sublethal doses actually stimulated patulin production by A. ochraceus 3174 1000 NG this mold (33). Yousef and Marth (186) reported in 1981 aNG = no growth. that sublethal amounts of sorbate sometimes resulted in bFrom Liewen and Marth (93). greater production of aflatoxin by A. parasiticus than

JOURNAL OF FOOD PROTECTION, VOL. 48, APRIL 1985 SORBIC ACID INHIBITS MICROBIAL GROWTH 369

charomyces acidifaciens and S. balii to the antimycotic agent independent of the acid used to acidify the growth medium. At 0.1% potassium sorbate, growth of all four yeast strains was completely inhibited. Effects of potas­ sium sorbate at pH 5.0 on growth of S. rouxii are shown in Fig. 3. While growth was rapid in the absence of pre­ servative, at 0.05% potassium sorbate growth was slowed and at 0.1% cell death occurred. Some strains of S. rouxii can acquire resistance to sorbate. Bills et al. (22) showed that a strain of S. rouxii preconditioned in 0.1% sorbate showed an increased resistance to sorbate when inoculated into a growth medium or chocolate syrup. Beech and Carr (13) found strains of S. cerevisiae, S.

rouxii, Saccharomyces carlsbergensis, (Saccharomyces Downloaded from http://meridian.allenpress.com/jfp/article-pdf/48/4/364/1657271/0362-028x-48_4_364.pdf by guest on 29 September 2021 3 4 uvarum), Torulopsis colliculosa, Brettanomyces bruxel- TIME(DAYS) lerisis, C. utilis and Triganopsis varabilis that were resis­ Figure 2. Viability of conidia of Penicillium roqueforti when tant to 0.1% sorbic acid in a wort medium at pH 5.4. exposed to various sorbic acid solutions at pH 4.75. From Sorbates inhibit growth of yeasts in cucumber fermen­ Liewen and Marth (92). tations (3, 24, 42, 43, 44, 124, 155), on the surface of hams (9), on high-moisture dried fruits (23, 115), in cot­ when media were free of sorbate. Similar results have tage cheese (27, 73), on smoked fish (26, 62) and in been reported by Bauer et al. (12) and Tsai et al. (174). fruit juices (15, 58, 121). Yousef and Marth (187) demonstrated that sorbate inhi­ bited production of aflatoxin in resting cultures of A. EFFECTS OF SORBATE ON GROWTH OF BACTERIA parasiticus and concluded that sorbate inhibited aflatoxin biosynthesis by inhibiting transfer of substances from the Information on the effect of sorbate against bacteria is growth medium into the cell. not nearly as comprehensive as it is for yeasts and molds. Some reports suggest that sorbate exerts a selective inhib­ EFFECTS OF SORBATE ON GROWTH OF YEASTS ition against all catalase-positive microorganisms and can be used as a selective agent for catalase-negative lactic Sorbates are generally very effective in controlling acid bacteria and Clostridia (57, 124, 176, 181, 182). spoilage yeasts in foods, although they cannot be used However, these conclusions may have been incomplete. directly in yeast-leavened products because of inhibition In some instances, pH of the medium was not controlled of baker's yeast (36). In broth culture at pH 4.5, Bell and hence the active undissociative form of sorbic acid et al. (14) found 0.1% sorbic acid to be inhibitory to may not have been present in amounts sufficiently 32 species of yeasts. large for effectiveness. The effectiveness of sorbate in Since yeasts do not compete effectively with most bac­ these studies was dependent upon factors such as concen­ teria in neutral to slightly acidic food products with high tration of the compound, laboratory medium used, pH of aw (0.98-0.995), this group of microorganisms has been the medium and type and strain of microorganism of minor importance in the spoilage of most food prod­ examined (161, 162). Hansen and Appleman (69) re- ucts. However, under certain storage conditions (low pH, intermediate aw, temperature slightly below room temper­ ature) a product may be susceptible to spoilage by yeast (141). Salad dressings and related products are sometimes spoiled by yeasts (159). Saccharomyces balii, because of its tolerance of acidic conditions, a high osmotic pressure and preservatives such as sorbate, is a universally recog­ nized problem in the salad dressing industry (126, 127, 159). Other products subject to spoilage by osmotolerant yeasts include carbonated beverages, cordials, tomato sauce, honey, maple syrup, molasses, some candy, jams, jellies and chocolate syrup (141). Under certain condi­ tions sorbates are effective in controlling spoilage yeasts. Restaino et al. (141) studied the effects of acids com­ bined with sorbate on growth of four osmophilic yeasts. Figure 3. The effects of potassium sorbate on the growth of At 0.05% potassium sorbate (pH 5.0), growth of all four S. rouxii, in supplementedipplemented potatopotato dextrose broth, pH 5.0. strains was slowed, although Saccharomyces rouxii and O^ = control. O = 500 ppm sorbate.sorbate. A = 7000 ppm sorbate. Saccharomyces bisporus were more resistant than Sac­ Adapted from Restaino et al. (129)

JOURNAL OF FOOD PROTECTION, VOL. 48, APRIL 1985 370 LIEWEN AND MARTH ported that sorbic acid neither inhibited nor stimulated perfringens and S. aureus. Several other reports have in­ growth of Clostridia in laboratory media at pH 6.7. Ham- dicated that sorbate or sorbate-nitrite combinations are ef­ den et al. (68) found that catalase-negative yogurt starter fective inhibitors of C. botulinum (69,70,74, bacteria were inhibited by 0.1% sorbic acid. Costilow et 78,100,112,113,147,162). al. (43) found two strains of catalase-positive Pediococ- cus cerevisiae which were as tolerant to sorbate as were RESISTANCE TO AND METABOLISM OF SORBATE BY catalase-negative bacteria. MICROORGANISMS Doell (54) found that 0.75% sorbic acid at pH 5.0 in­ activated salmonellae and E. coli in 48 h at 37°C. Con­ Some microorganisms can grow in the presence of centrations as low as 0.075% were bacteriostatic. Park large amounts of sorbate, and some actually metabolize and Marth (779) were among the first in the U.S. to de­ or degrade it. Melnick et al. (705) reported in 1954 that finitively demonstrate the effectiveness of the undis- high initial mold populations degraded sorbic acid in sociated sorbic acid against bacteria. They found that cheese. They hypothesized that the degradation occurred

0.3% sorbic acid at pH 5.0 inactivated S. typhimurium as a result of (3-oxidation with C02 and H20 as end-Downloaded from http://meridian.allenpress.com/jfp/article-pdf/48/4/364/1657271/0362-028x-48_4_364.pdf by guest on 29 September 2021 in 12 h in nutrient broth at 37°C. Complete inactivation products. Marth et al. (707), in 1966, were the first in­ in evaporated milk (pH 5.0) at 7°C required 55 d. Reduc­ vestigators to find that some molds in the genus Penicil- ing sorbic acid concentration or raising the pH of the sub­ lium were able to degrade sorbate to 1, 3-pentadiene, a strate increased the time needed for inactivation. Growth volatile compound with an extremely strong kerosene-like occurred in nutrient broth and skim milk with 0.2 and odor. They proposed that 1, 3-pentadiene was produced 0.3% sorbic acid when the pH was not adjusted. Inactiva­ as a result of a decarboxylation reaction, which elimi­ tion of S. typhimurium was more rapid in cold-pack nated inhibitory sorbate from the growth medium and al­ cheese food that contained sorbic acid rather than sodium lowed growth of the mold. Later, presence of 1, 3-pen­ propionate or no antimycotic agent (720). tadiene, probably because of microbial activity, was also Growth of two strains of Pseudomonas fluorescens was observed in a sorbate-treated noncarbonated beverage (63) inhibited by 0.05% sorbate in trypticase soy broth (pH and in sorbate-treated Feta cheese (77). 5.5) and 0.20% sorbate delayed growth of both strains Kurogochi et al. (85,86) reported that sorbic acid was at pH 6.0 (142). P. putrefaciens was inactivated by metabolized by Mucor species to 4-hexenol and by Geo- 0.20% sorbate in trypticase soy broth at pH 6.0 (144). trichum species to 4-hexenoic acid and ethyl sorbate. P. fragii is also inhibited by sorbate (770). Tahara et al. (765) reported that all Mucor species they Growth of S. aureus in brain heart infusion (BHI) at tested metabolized sorbic acid to 4-hexenol. Takanami et pH 5.0 was inhibited by 1% sorbate, but at pH 7.0 the al. (766) found a decrease in sorbic acid in organism grew in the presence of 5% sorbate (88). Potas­ "Yamagobozuke", a pickled vegetable, which was as­ sium sorbate (0.25%) had no effect on the growth of S. sociated with an increase in 4-hexenoic acid. The aureus in BHI at pH 6.0 (118). In pasteurized minced metabolite was presumably formed during the lactic fer­ cod inoculated with S. aureus, 0.5% sorbate resulted in mentation of the vegetable. Losses of sorbate have been a markedly slower rate of growth of the pathogen as well noted in cucumber fermentation, and may also be the re­ as a delay in enterotoxin production compared to when sult of lactic fermentation (3). sorbate was absent (98). Bacon containing 0.26% potas­ Raduchev and Rizvanov (133) also reported that some sium sorbate did not support growth of S. aureus (125). microorganisms, including Lactobacillus plantarum, Growth of S. aureus in pie fillings that are otherwise ex­ Streptococcus lactis, Acetobacter species, and penicillia cellent microbiological media can be prevented by adding could use sorbic acid as a carbon source. Lukas (7,9,96) sorbate (729, 752). reported that A. niger and molds in the genus Penicillium Trypticase soy broth with 0.1 or 0.2% sorbate adjusted could decompose sorbic acid. It was shown that sorbic to pH 5.5 was bacteriostatic to salmonellae and Yersinia acid was not stored in the cells or decomposed by ex­ enterocolitica and extended the lag phase of P. fluores­ tracellular enzymes. It was probably taken up into the cens for 24 h (128). A reduction in numbers of S. cells and metabolized to C02 and H20. Rehm et al. typhimurium was noted in cooked turkey meat treated (139) reported that A. niger and P. fluorescens were able with 0.1% sorbate. Growth of Vibrio parahaemolyticus to metabolize sublethal concentrations of sorbic acid. was inhibited by 0.1% sorbic acid in crab meat homoge- Lawrence (89,90) showed that spores of P. roqueforti nates (145). Sorbate reduced the number or delayed were able to oxidize fatty acids to methyl ketones. growth of spoilage bacteria in cottage cheese (27, 41), Wines treated with sorbic acid to prevent yeast spoil­ poultry (47, 73, 109, 143, 146, 147, 149, 167, 168), age often develop a geranium-like odor (75,79). Crowell frankfurters (67), beef (66, 67), seafood (4, 37, 62, 98, and Guymon (46) identified ethyl sorbate; 2, 4-hexadien- 163) and yogurt (68); sorbate inhibited Bacillus cereus l-ol; l-efhoxyhexa-2, 4-diene; 3, 5-hexadien-2-al; and 2- and B. subtilis in rice filling of Karelian pastry (134). ethoxyhexa-3, 5-diene in a sorbate-treated red table wine Tompkin et al. (769) found that adding 0.1% potas­ spoiled by lactic acid bacteria. The latter compound was sium sorbate to cooked uncured sausage retarded growth responsible for the geranium-like odor and was thought and toxin production by Clostridium botulinum. The to result from the lactic fermentation. Wurdig et al. (180) treatment also delayed or retarded growth of Clostridium studied the geranium-like odor in wines and concluded

JOURNAL OF FOOD PROTECTION. VOL. 48, APRIL 1985 SORBIC ACID INHIBITS MICROBIAL GROWTH 371 that it was a result of degradation of sorbic acid to 2, SUMMARY 4 hexadien-1-ol. The geranium-like odor has also been reported by Burkhardt (35) and Radler (132), both of While sorbic acid is among the most commonly used whom attributed it to degradation of sorbic acid by lactic of the antimicrobial food preservatives, it does have limi­ acid bacteria. tations in its effectiveness. The maximum permissible There have been numerous reports of sorbate-resistant dosage in most foods is 0.3%. At this concentration, sor­ yeasts (22,77,126,127,178), but none of these has indi­ bic acid is effective against a variety of microorganisms; cated that degradation of sorbate occurs. Warth (178) however, some grow at concentrations much greater than found that resistance to preservatives by S. bailii resulted 0.3%. Some molds and bacteria are able to degrade sor­ primarily from an inducible, energy-requiring system bic acid. Environmental factors such as pH, water activ­ which transports preservatives from the cell. ity, temperature, atmosphere, microbial load, type of microbial flora, and certain food components can influ­ ence the effectiveness of sorbic acid. All of these factors

should be considered when using sorbic acid as an anti­ Downloaded from http://meridian.allenpress.com/jfp/article-pdf/48/4/364/1657271/0362-028x-48_4_364.pdf by guest on 29 September 2021 MECHANISM OF INHIBITION BY SORBATE microbial preservative. Each situation should be consid­ ered on an individual basis and environmental factors The process by which sorbate inhibits microbial growth should be controlled to give optimal antimicrobial activ­ is not clear and defined. Some work has implicated inhib­ ity. ition of various enzyme systems. In 1954, Melnick et al. (105) proposed that sorbic acid inhibits certain dehyd­ ACKNOWLEDGMENT rogenase enzymes which are involved in the (3-oxidation of fatty acids. Other workers have postulated that sorbate A contribution from the College of Agricultural and Life Sciences, University of Wisconsin-Madison. may inhibit respiration through a competitive action with acetate at the site of acetyl-CoA formation (70). 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