A CETIC A CID

Antimicrobial Properties

Acetobacter and heterofermentative lactic acid bacteria (heterolactics) produce acetic acid as a by- product of their metabolism and, as such, are more tolerant to this acid than homofermentative lactic acid bacteria (homolactics), which do not. These organisms are found naturally associated with fermented products, such as pickles, sauerkraut, and vinegar.

Adams and Hall (1988) showed a weakly synergistic inhibitory effect between acetic and lactic acids with Salmonella enteritidis and Escherichia coli. Although these acid mixtures might be expected to occur naturally during fermentations by heterolactic bacteria, this apparent synergy has not been demonstrated in other foods. This association could explain the apparent stability that occurs in natural fermentations, such as sauerkraut. Sauerkraut manufacture is a fermentation process brought about by a natural succession of microbes that inhabit the surface of cabbage leaves. The succession typically begins with growth of coliforms and natural microflora followed by Leuconostoc mesenteroides (heterolactic), then by Lactobacillus plantarum (homolactic), a more acid-tolerant organism. McDonald et al. (1990) found that growth of L. mesenteroides ceased when the internal pH of the cells reached 5.4 to 5.7, whereas growth stopped in L. plantarum at pH 4.6 to 4.8. L. plantarum maintained its pH gradient in the presence of either 160 mM sodium acetate (SA) or sodium lactate down to an external pH of 3.0.

Microorganisms vary in their susceptibility to acetic acid. Concentrations of acid lower than those needed to inhibit Saccharomyces cerevisiae (pH 3.9) and Aspergillus niger (pH 4.1) (Levine and Fellers, 1939) inhibited Bacillus cereus (pH 4.9), Salmonella Aertrycke (pH 4.9), and Staphy- lococcus aureus (pH 5.0). Bacillus and Clostridium species and Gram-negative bacteria were more inhibited at pH 6.0 than lactic acid bacteria, Gram-positive bacteria, yeasts, and molds; however, as the pH decreased to 4.0, all of the groups were similarly affected (Woolford, 1975b). Owen (1946) suggested that acetic acid inhibited Gram-positive organisms.

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S. aureus was most sensitive to acetic, followed by citric, lactic, malic, tartaric, and hydrochloric acids (Nunheimer and Fabian, 1940). Acetic acid exerted the most inhibition but had the lowest dissociation constant, whereas tartaric acid had one of the highest dissociation constants yet exerted the weakest inhibitory action. Similar results were noted when various acids were compared at the pH level required for a 90% and 99% reduction in numbers of S. aureus in 12 hours (Minor and Marth, 1970). Acetic acid was effective at pH 5.2 and 5.0, respectively, followed by lactic, pH 4.9 and 4.6; citric, pH 4.7 and 4.5; and HCl, pH 4.6 and 4.0. Both studies concluded that the anti- microbial effect was the result of the undissociated molecule. S. aureus preconditioned in acetate buffer (pH 4.4 to 4.6) at 20°C shifted to pH 7.0 buffer, and exposed to 40°C became injured, but no injury occurred in cells not exposed to acidic conditions before heating (Smith et al., 1984).

B. cereus was inhibited at pH 5.0 with 0.15 M acetic acid and with 0.33 M lactic acid (Wong and Chen, 1988). A 0.1-M concentration of acetate and lactate completely inhibited multiplication at pH 6.1 and 5.6, respectively, and caused 50% inhibition of spore germination at pH 4.2 and 4.3, respectively.

Buffered acidulant systems (brain heart infusion [BHI] broth, pH 4.8, 22°C) containing 1.0% acetic acid and 1.0% lactic, malic, tartaric, or citric acid were far more effective in reducing numbers of S. aureus, Salmonella Blockley, Streptococcus faecalis, and E. coli than unbuffered systems. This finding is important in the manufacture of medium acid foods containing more than one organic acid, such as mayonnaise (Debevere, 1988).

E. coli O157:H7 is considered more acid tolerant than strains of E. coli. When E. coli O157:H7 was inoculated into tryptic soy broth (TSB) containing acetic, citric, lactic, malic, and tartaric acids, growth occurred at pH 5.5 for all acidified media at 10°C except for acetic acid. At 25°C and 37°C, E. coli O157:H7 grew in all media at pH 5.0 with scant growth in TSB containing acetic acid. Malic acid allowed growth at pH 4.5, and tartaric acid permitted growth at pH 4.0 (Conner and Kotrola, 1995; Conner et al., 1997).

Traditionally acidic foods contain a single or multiple acids, added or developed through fermentation. In addition, other ingredients can interact with acid to influence the degree of inhibitory action. Reduced-calorie mayonnaise (RCM) manufactured with 0.1%, 0.3%, 0.5%, or 0.7% acetic acid in the aqueous phase and adjusted to pH 4.0 with hydrochloric acid was compared to cholesterol-free (egg yolk-free) reduced-calorie mayonnaise (CFM) manufactured with 0.3% and 0.7% acetic acid (Glass and Doyle, 1991). Both formulations were inoculated with approxi- mately 108 _{CFU/ml of an 8-strain mixture of Salmonella or a 6-strain cocktail of Listeria mono-}

cytogenes and held at 23.9°C. Salmonella were inactivated within 48 hours in both RCM and CFM formulated with 0.7% acetic acid with reduction to undetectable levels in 1 and 2 weeks with 0.5% and 0.3% acetic acid formulations, respectively. Destruction of Salmonella occurred more quickly in CFM than RCM, most likely because of the absence of egg yolks in CFM and the presence of egg white, which has recognized antimicrobial properties. L. monocytogenes, although more resis- tant than Salmonella, was reduced by 4 logsin both mayonnaises within 72 hours in formulations containing 0.7% acetic acid. L. monocytogenes was also reduced to undetectable levels in RCM within 10 days and in CFM within 14 days compared to 2 days for Salmonella. It is unlikely that these products would contain such high initial levels of either of these organisms, especially with the use of pasteurized egg products, therefore the standard use of 0.7% acetic acid, pH 4.0, in the aqueous phase of CFM and RCM would still produce a microbiologically sound product.

A concentration of 0.4% to 0.8% reduced growth of Micrococcus and Bacillus species, some strains of S. aureus, Gram-negative aerobic bacteria, and Enterobacteriaceae in Japanese fermented soy sauce (Hayashi et al., 1979). An increased toxic effect of acetic acid against Saccharomyces rouxii and Torulopsis versatilis was demonstrated in brine fermentation of soy sauce as the pH decreased from 5.5 to 3.5 (Noda et al., 1982).

The effect of temperature-acid interactions is noteworthy for the psychrotrophic pathogens, L. monocytogenes and Yersinia enterocolitica. Acetic acid caused greater inactivation of L. mono- cytogenes than lactic and citric acids and increased inhibition as the temperature of incubation

decreased from 35°C to 13°C; concentration ≥0.3% inhibited L. monocytogenes irrespective of temperature. However, citric acid caused greater injury than either lactic or acetic acids and injured organisms survived longer at low temperatures (Ahamad and Marth, 1989, 1990).

Acetic acid displayed the greatest antimicrobial action against L. monocytogenes when com- pared to citric, malic, lactic, and hydrochloric acids at equal pH values and at all incubation times and temperatures (Sorrells et al., 1989). When comparisons were based on equal molar concentra- tions, however, citric acid was more bactericidal than other acids at 25°C and 35°C, but malic acid was more bacteriostatic than other acids at 10°C. The greatest antimicrobial effect occurred at 35°C with the greatest survival at 10°C. The minimum inhibitory pH level was pH 4.4 for malic, citric, and hydrochloric acids, pH 4.4 to 4.6 for lactic acid, and pH 4.8 to 5.0 for acetic acid. Sorrells and Enigl (1990) also demonstrated interactive effects between sodium chloride, acidulants, and tem- perature.

Similar growth inhibition was demonstrated for Y. enterocolitica at lower pH levels as the temperature increased. Acetic acid was more effective than lactic or citric acids in producing this effect (Adams et al., 1991; Brocklehurst and Lund, 1990; Karapinar and Gönül, 1992a; Little et al., 1992). Conversely, injury and death rates were not affected by refrigeration temperatures for Salmonella Bareilly, a mesophilic organism. A 90% reduction of the population required exposure to 0.01 N for 75 min, 0.05 N for 8.5 min, 0.10 N for 5 min, and 0.2 N for 4 min. Acid injury did not appear to involve damage to ribosomes or other nucleic acid material, but recovery required the presence of amino acids or peptones (Blankenship, 1981).

Raw produce is now being subjected to acid rinses in some production practices as a means of reducing high levels of spoilage organisms and foodborne pathogens. Because produce is typically refrigerated with only minimal processing, these foods can be the carriers of psy- chrotrophic pathogens, such as Y. enterocolitica. Parsley, inoculated with approximately 107 _CFU/g

Y. enterocolitica, was dipped in acetic acid solutions (1%, 2%, or 5%) or vinegar (30%, 40%, or 50%) for 15 or 30 minutes. Y. enterocolitica was not detectable after exposure to 2% and 5% acetic acid or 40% and 50% vinegar for 30 minutes (Karapinar and Gönül, 1992b).

Acetic acid applied as a volatile compound (1000 mg/L of air) for 7 hours at 60°C achieved >3 log reduction of six Salmonella strains inoculated onto alfalfa seeds compared to 1.9 log reduction for the nontreated seeds (Weissinger et al., 2001). Exposure at 50°C for 12 hours with lower levels (100 and 300 mg/L of air) reduced the population >1.7 log without affecting germi- nation of seeds. Treatment at low temperature (4 days at 10°C) with 200 and 500 mg/L of air was effective in reducing Salmonella by 2.33 and 5.72 logs, respectively, but resulted in changes in sensory characteristics. Mung bean seeds inoculated with 3 to 5 logs of S. Typhimurium, E. coli O157:H7, and L. monocytogenes were exposed to acetic acid at a concentration of 242 µL/L of air for 12 hours at 45°C. This treatment reduced S. Typhimurium and E. coli O157:H7 to nondetectable levels by enrichment methods, but L. monocytogenes was isolated from 2 of 10, 25-g samples. In addition, vapor treatment also reduced levels of resident microflora on seed but did not affect seed germination rates (Delaquis et al., 1999). Immersion of alfalfa seeds in mixtures containing 5% lactic or citric acid for 10 minutes resulted in several log reductions, but acetic acid was less effective. In addition, seed germination was effected (Weissinger and Beuchat, 2000).

A variety of organic acid treatments has been used to control microbial loads associated with meat carcasses and products (Dickson and Anderson, 1992; Dorsa, 1997; Dorsa et al., 1997, 1998; Smulders and Greer, 1998). Numerous organic acid combinations, containing from 0.6% to 3.0% acetic acid at pH levels from 1.5 to 3.0 have been used to rinse pork, lamb, and beef carcasses or meat tissue. Treatments spanned varying amounts of time with concomitant decrease in microbial loads up to 3 logs. Depending on the treatments, there can be adverse sensory effects including discoloration of meat, off-odors, and off-flavors.

Pork carcasses inoculated with 106 _{CFU/ml S. Enteritidis were sprayed with acetic acid at pH}

1.5 or 2.0 for 30 or 60 seconds (Biemuller et al., 1973). Total plate counts were reduced by 4 logs at pH 1.5, a twofold reduction at pH 2.0, 1 log unit at pH 2.5, and no reduction in microbial

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numbers at pH 3.0. The treatment effectively reduced recovery of S. Enteritidis to 1 of 6 samples at pH 1.5 and 11 of 72 for pH 2.0. Although washing of pork carcasses with 1.5% acetic, citric, or lactic acid led to significant decreases in aerobic plate and coliform counts for acetic and citric acid washes after 14 days of storage at 2°C to 4°C, L. monocytogenes was detected in 69% of loins and 33% of chops, suggesting that acids had limited effectiveness (Fu et al., 1994). Increasing the concentration to 2% acetic acid or using 200-ppm hypochlorite solutions before vacuum packaging and storage for 28 days at 4°C significantly lowered aerobic, anaerobic, and lactic acid bacteria counts, however, some discoloration occurred (Cacciarelli et al., 1983). A 3% concentration of acetic acid was quite effective in reducing counts of Enterobacteriaceae in vacuum-packaged pork stored for 6 weeks at 2°C to 4°C (Mendonca et al., 1989b). An application of 2% acetic acid reduced the incidence of Salmonella on pork cheek meat in addition to significantly reducing aerobic plate and coliform counts (Frederick et al., 1994). An 18% acetic acid or 12% lactic acid spray signifi- cantly reduced bacterial counts on lamb carcasses. At this concentration, the pH level decreased to 2.1 and 1.5, respectively, and bleaching of the carcasses occurred (Ockerman et al., 1974).

As part of recently established guidelines by the U.S. Department of Agriculture Food Safety Inspection Service (FSIS), organic acids can be used in preevisceration rinse systems consisting of a water rinse followed by an organic acid rinse. Application of a 3% concentration of acetic acid as a spray on beef carcasses was not significantly better than water washes in reducing levels of E. coli O157:H7; however, a final application of acid washes could provide some residual effect during storage (Brackett et al., 1994; Cutter et al., 1997; Cutter and Siragusa, 1994; Dorsa et al., 1997; Hardin et al., 1995).

Quartey-Papafio et al. (1980), found that a 1% formic and 1% acetic acid combination exposed to beef for up to 20 minutes was effective in reducing a variety of bacterial species. Beef cubes treated with 1.2% acetic acid or 0.6% acetic and 0.046% formic acids for 10 seconds caused discoloration from both treatments, but beef treated with the mixture of acids did not differ in flavor from untreated beef (Bell et al., 1986). Hamby et al. (1987) used intermittent sprays of 1% acetic acid or lactic acids, which resulted in significant reductions in aerobic plate count (1.8 to 4.3 log/cm2_{). Meat was sprayed, vacuum packaged, and stored for 28 days at 2°C in a high-oxygen}

barrier film. Lactobacillus species predominated as storage progressed. Application of a 2% lactic or 2% acetic acid spray to beef strip loins and stored at 1°C resulted in significantly lower aerobic and lactic acid bacteria counts over 112 days of storage (Goddard et al., 1996).

Reductions in microbial counts depend on the water temperature, type of chemicals, and application rate and sequence in the process (Gorman et al., 1995). Anderson et al. (1977) reduced bacterial counts by 99.6% on meat using a 3% concentration of acetic acid before washing. They found that acetic acid was a superior sanitizer compared to hypochlorite and had a greater residual effect. Further work by Anderson and Marshall (1989) demonstrated that application of acetic acid at 70°C was most effective in sanitizing beef semitendinous muscle inoculated with E. coli, S. Typhimurium, or manure slurry. Temperature played a greater role in reducing numbers than did the acid concentration. Microbial loads on beef carcasses subjected to machine washing and sanitization with 1.5% acetic acid were reduced more when treatments were conducted at 52°C than at 14.4°C (Anderson et al., 1987). Incorporation of pulsed power electricity and a 2% acetic acid spray led to improved reduction of inoculated E. coli O157:H7 and S. Typhimurium on beefsteaks (Tinney et al., 1997).

Dickson (1992) contaminated lean and beef tissue surfaces with S. Typhimurium followed by treatment with 2% acetic acid. S. Typhimurium was reduced by 0.5 to 0.8 log CFU/cm2_{; however,}

this was not significantly different from the controls. S. Typhimurium reductions were greater when the bacteria were attached to fatty tissue, possibly because this tissue retained less moisture (20%) than lean tissue (75%) and the reduced water activity could have enhanced the antimicrobial effects. It was noted that the use of acetic acid as a rinse for beef tissue led to sublethal injury of bacterial cells. An increase in organic material, such as rumen fluid, dirt, or manure, resulted in less effective reduction of S. Typhimurium. Unda et al. (1991) inoculated beef roasts by injection and on the

surface with approximately 103 _{CFU of Clostridium sporogenes and L. monocytogenes. The acetic}

acid in the brine inhibited anaerobic and aerobic bacteria.

Intervention steps incorporated into poultry processing have been effective in reducing microbial contamination. Poultry scald water containing 0.1% acetic acid at 52°C decreased levels of S. Typhimurium and Campylobacter jejuni by 0.5 to 1.5 logs (Okrend et al., 1986). Addition of 1.0% acetic acid caused instantaneous death. Levels of 0.2% to 0.5% acetic acid reduced Entero- bacteriaceae counts by 2.24 log (Lillard et al., 1987). Because of the possible attachment of Salmonella to broiler chicken skin, greater than a 4% concentration of acid was needed to reduce levels by 2 logs (Tamblyn and Conner, 1997). Despite these treatments, further poultry processing can reintroduce microorganisms onto carcasses.

Acidification of the chill water at the end of processing delivers a second intervention step to reduce microbial loads and to increase the shelf life of poultry. Adjustment of the chill water to pH 2.5 with acetic acid was the most effective antimicrobial treatment followed by other acids in descending order of effectiveness: adipic, succinic, citric, fumaric, and lactic (Mountney and O’Malley, 1965). Despite the reduction in microbial numbers at this pH, acetic acid caused the skin of the poultry to be hard and leathery.

Other studies continued to explore the use of acetic acid in chill water, but changes in pH and concentration of acid were more successful. Immersion of broilers for 10 minutes in 0.6% acetic solution acid (pH 3.0) did not result in a significant reduction of aerobic plate counts but did significantly reduce levels of Enterobacteriaceae by 0.71 log most probable number (MPN)/ml (Dickens et al., 1994). Expanding on their work, Dickens and Whittemore (1994) exposed broilers to 0.3% and 0.6% acetic acid under the same conditions, with and without the use of air injection to agitate the chill water. Again, aerobic plate counts were unaffected by the treatments, but Enterobacteriaceae counts were significantly reduced by 0.86 log MPN/ml for the 0.3% acid and 2.35 log MPN/ml for the 0.6% acid solutions. Air injection did not affect reduction of these counts. There was no significant difference in texture or sensory characteristics between the treatments, although the skin of the 0.6% acetic acid-treated carcasses was darkened or yellowed (Dickens et al., 1994). Water pockets occurred under the skin of chicken carcasses with air-agitated samples (Dickens and Whittemore, 1994).

Products are now being preserved by a number of antimicrobial compounds using a multiple- barrier approach to preservation. Kurita and Koike (1983) combined 3% ethanol, 0.05% acetic acid, 1 mM perillaldehyde—or 0.5%, 0.05%, and 0.5 mM, respectively, of the same components—with 2% salt to inhibit growth of contaminating microorganisms for more than 20 days at 27°C. Rubin (1978) found that acetic and lactic acid acted synergistically to retard growth of S. Typhimurium using a model system. El-Shenawy and Marth (1989b) investigated the effect of acetic, citric, lactic, and tartaric acid adjusted to pH 5.6 or 5.0 on inhibition of L. monocytogenes. Acidified media showed greater inhibitory activity at 35°C than at 13°C. Acetic and tartaric acids were more inhibitory than lactic and citric acids.

The acid level could be reduced when used in combination with brines and sugar. Whole pickles preserved with brine at 12° Brix and 0.9% acetic acid did not support growth or toxin formation of Clostridium botulinum (Ito et al., 1976). Stroup et al. (1985) found that mushrooms reached an equilibrium pH of 4.6 depending on the concentration of acid in can brine within 1 day with 1.0% acetic acid compared to 2 days for 0.7% citric acid. For pearl onions, a 0.35% solution required 7 days for equilibration for acetic and citric acid but 1 day for 0.7% acetic and 4 days for the same concentration of citric acid.

Nisin (an approved bacteriocin for some foods; see Chapter 7) coupled with 0.05% and 0.5% concentrations of acetic, lactic, or citric acids or glucono-delta lactone were studied using Bacillus spores heated at pasteurization temperatures of 65°C and 95°C and recovered at 12°C, 20°C, and 30°C (Oscroft et al., 1990). Although each individual acid influenced the heat resistance of Bacillus spores differently, acetic acid was the most destructive acid at all combinations of parameters. As the incubation temperature to recover spores was lowered, germination and outgrowth of Bacillus

Organic Acids 99

spores were more restricted. Nisin, in combination with organic acids, displayed synergistic effects in increasing the destruction of the organism. It was found that acetic acid was the most effective acid, followed by glucono-delta lactone, lactic, and citric acid when spores were heated at 95°C for 15 minutes and acetic, lactic, glucono-delta lactone, and citric acids when temperatures were reduced to 65°C for 60 minutes. Foods, particularly precooked, chilled, ready-to-eat products, are especially susceptible to spoilage because of indigenous microbial populations. A combination of these parameters could be used to extend the shelf life of these products.

Although acetic acid is generally used as an inhibitor of bacteria and yeasts, it is effective against the black bread molds, A. niger, and Rhizopus nigrificans at pH 3.5. Aspergillus fumigatus required 0.4% acetic acid at pH 5.8 and 0.2% acid at pH 5.0 and below to inhibit growth (Kirby et al., 1937). When used as a surface application, a 1% concentration of acetic acid at pH 4.5 completely inhibited growth and aflatoxin production by Aspergillus parasiticus. Lower concen- trations of 0.6% or 0.8% partially inhibited growth and decreased toxin formation by 70% and 90%, respectively (Buchanan and Ayres, 1976). Cruess and Irish (1932) showed that apple juice containing 0.8% to 1.0% acetic acid adjusted to pH 3.5 inhibited Saccharomyces ellipsoideus and Penicillium glaucum, but a concentration greater than 4% was needed when the pH was at 7.0. Populations of Pseudomonas aeruginosa were reduced by 99.999% after exposure to a 1.0% concentration of acetic acid, suggesting its use as a decontamination agent for equipment (Hedberg and Miller, 1969).

Other forms of acetic acid have antimicrobial uses. Peracetic acid is formed by an oxygen molecule bound to the carboxyl carbon atom of acetic acid. On contact with organic substrates, it