L ACTIC A CID

Antimicrobial Properties

Lactic acid has been used more extensively for its sensory qualities than its antimicrobial properties in the past, although more recently this acid has been used as a rinse for beef, pork, and chicken carcasses. The inhibitory capacity of this acid lies in its reduction of pH to levels below which bacteria cannot initiate growth. In fermented foods, lactic acid coupled with other antigrowth factors excreted by lactic acid-producing microorganisms inhibits competing microorganisms.

Lactic acid was an excellent inhibitor of spore-forming bacteria at pH 5.0 but was ineffective against yeasts and molds (Woolford, 1975a). Lactic acid was about four times more effective than malic, citric, propionic, and acetic acid in limiting growth of Bacillus coagulans, the organism responsible for flat-sour spoilage in tomato juice (Rice and Pederson, 1954). Lactic acid was more inhibitory to Mycobacterium tuberculosis as the pH decreased (Dubos, 1950). Gill and Newton (1982) suggested that the inhibitory effect for psychrotrophic organisms was related to a decrease in pH rather than degree of dissociation. Cold-pack cheese normally prepared with lactic acid was reformulated with acetic acid and inoculated with S. Typhimurium. Destruction rates for S. Typh- imurium were similar for both acids (Park et al., 1970).

Lactic acid sprays have been effective in limiting microbial growth on meat carcasses under a variety of storage conditions. A 1.0% to 1.25% concentration of acid sprayed on veal carcasses, followed by vacuum packaging, lowered microbial counts after storage for 14 days at 2°C, but a 2% concentration led to discoloration of the carcass surface (Smulders and Woolthuis, 1983; Woolthuis and Smulders, 1985). A 2% L-lactic acid treatment at pH 2.3 of veal tongues, combined

with vacuum packaging and storage at 3°C, reduced aerobic mesophilic plate counts from 5.6 to 2.7 log CFU/cm2 _{(Visser et al., 1988). Optimal decontamination was achieved when 1% lactic acid}

solution at 55°C was sprayed on beef carcasses immediately after dehiding and after evisceration (Prasai et al., 1991). Osthold et al. (1984) found that spraying beef and sheep carcasses with a combination of 1% lactic, 2% acetic, 0.25% citric, and 0.1% ascorbic acids selectively inhibited Enterobacteriaceae at 10°C.

Similar reductions in microbial counts occurred with a 1.25% lactic acid spray of beef carcasses followed by a treatment of hot-boned cuts with 2% acid, vacuum packaging, and storage for 10 days. Acid treatment when combined with vacuum packaging was more effective in prolonging shelf life than vacuum packaging alone (Smulders and Woolthuis, 1985). Enterobacteriaceae contaminated 50% of the untreated samples, which was reduced to 10% after treatment. A study using lactic acid sprays of skinned cow heads found that 1% acid was effective in significantly reducing total counts and extending shelf life by 3 days at 4°C and 1 day at 15°C and 20°C (Cudjoe, 1988). Because most other studies chose refrigeration storage temperatures, this study showed that lactic acid was effective at higher temperatures as well.

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Hot-boned, sub-primal meat was injected with one of three treatments: 0.3 M calcium chloride solution (CAL, pH 5.1), 0.3 M lactic acid solution (LAC, pH 3.0), or a combination (1:1) CAL/LAC (COM, pH 2.5) at a volume equal to 10% of the initial sub-primal weight. Significantly higher aerobic plate counts were obtained from hot-boned cuts over 17 days postmortem compared to cold-boned cuts. Aerobic plate counts of meats injected with CAL were significantly higher than with LAC or COM. Furthermore, COM improved tenderness in muscle cuts, but control cuts retained more desirable flavor profile (Eilers et al., 1994).

Kotula and Thelappurate (1994) compared acetic acid and lactic acid solutions applied at two concentrations, 0.6% or 1.2%, and at two time intervals, 20 or 120 seconds, at 1°C to 2°C as a dip for rib eye steaks. Minimal reduction in total plate and E. coli counts was detected for beef dipped in acetic or lactic acid solutions compared with a water-dipped control at day 1. A residual effect was noted for the lactic acid-treated tissue in that microbial counts were significantly decreased compared to the control steaks after 9 days of storage, but this effect was not seen with acetic acid- treated tissue. Acid-treated samples were lighter in color as a result of leaching of the pigment during immersion, but shear values, moisture content, and sensory analysis were not affected by acid treatment.

Postchill beef rounds inoculated with E. coli O157:H7 or S. Typhimurium were subjected to an automated wash water system (Castillo et al., 1998; 2001a,b). The system alone produced a 3- log reduction. Sprays containing 4% lactic acid applied at 55°C for 30 seconds or at 65°C for 15 or 30 seconds led to unrecoverable levels of E. coli. When sequential treatments were employed using pre-chill and postchill lactic acid sprays, E. coli was reduced by 6.8 to 7.2 logs. Microbial counts obtained from ground beef made from the treated beef rounds that were subjected to the multiple sprays were significantly lower than samples receiving a postchill rinse, only suggesting that lactic acid can exert a continued antibacterial effect during the shelf life of the product.

Beef trim meat used to make ground beef is of particular concern because of its higher microbial load. Beef trim was fabricated to make predominantly beef trim lean (BTL) and beef trim fat (BTF) products that were inoculated with bovine fecal samples (Kang et al., 2001a,b). The inoculated meat was subjected to sprays of tap water (15°C to 17°C, 65 psi); 2% lactic acid (12°C to 15°C, 30 psi); hot water (30 psi) at 65°C, 71°C, 76°C, or 82°C; or hot air at 371°C, 426°C, 454°C, 482°C, or 510°C. In addition, combinations of the treatments were examined. All products were stored at 4°C and examined for aerobic, psychrotrophic, and presumptive lactic acid bacteria; total fecal coliforms; and E. coli biotype 1 counts. Greater reductions on all microbial loads were seen in BTF compared to BTL. Lactic acid application as the final step in a multiple-hurdle process led to a residual effect during storage for 7 days at 4°C. Similar work was conducted using lean pork trim (LPT) and fat-covered pork trim (FPT) (Castelo et al., 2001). As in beef trim, the FPT supported lower microbial numbers than LPT. Treatments containing lactic acid resulted in the lowest counts without affecting sensory qualities. Application of 95°C water alone or with 2% L-lactic acid was

effective in reducing E. coli O157:H7 and S. Typhimurium-inoculated beef trimmings. Meat color was affected after application and through storage at 4°C, but odor was not affected (Ellebracht et al., 1999).

Acuff et al. (1987) compared a combination of 1% lactic, 2% acetic, 0.25% citric, and 0.1% ascorbic acids adjusted to pH 2.6 with either 1% lactic acid at pH 2.9 or 1% acetic acid at pH 3.3. There was little difference in bactericidal activity between the combination and either acid alone on sub-primal cuts of beef. Dixon et al. (1987) confirmed their findings using beef strip loins stored in polyvinyl chloride (PVC) film for 6 days or high-oxygen barrier film for 28 days. When temperature changed from 20°C to 70°C or lactic and acetic acid concentrations increased individ- ually to 3%, aerobic bacterial counts and S. Typhimurium levels decreased approximately 1 log, <1 log for Enterobacteriaceae and 0.5 logs for E. coli (Anderson and Marshall, 1990a, b). The combination of acids performed as well as 1% lactic or acetic acids alone. Lactic acid was most effective against S. Typhimurium at 70°C, reducing numbers by 2 logs and Enterobacteriaceae by 1.5 logs (Anderson et al., 1992).

Lactic acid (2%), low-molecular-weight polylactic acid (2% PLA), and nisin (400 IU/ml) were used alone or in combination in beef to inhibit L. monocytogenes (Ariyapitipun et al., 2000). Beef was inoculated with L. monocytogenes before vacuum packaging and storage at 4°C. All treatments significantly reduced the number of L. monocytogenes; however, there was no significant difference between nisin, and low-molecular-weight polylactic acid, lactic acid, and polylactic acid. After 42 days of storage the initial inoculum of 5 logs was reduced to <1 log after using lactic acid, nisin, or nisin and lactic acid. Beef inoculated with E. coli O157:H7 was subjected to treatments of low- molecular-weight polylactic acid, lactic acid, and 200 IU/ml of nisin (Mustapha et al., 2002). After 28 days of storage, significant reductions were seen with all treatments, but no differences were seen between polylactic acid and lactic acid, and nisin did not enhance the effects of lactic acid against E. coli O157:H7. The use of these compounds was effective in reducing psychrotrophs and Enterobac- teriaceae (Ariyapitipun et al., 1999). Beef cubes were inoculated with L. monocytogenes and treated with 2% lactic acid, 40,000 IU/ml nisin, and 3200 units/ml pediocin. These compounds reduced the level of L. monocytogenes by 1.7, 1.1, and 0.6 log/6 cm2_{, respectively (El-Khateib et al., 1993).}

A novel approach to shelf life extension was the use of lactic or acetic acid-impregnated calcium alginate gels applied to the surface of beef tissue (Siragusa and Dickson, 1992, 1993). Although the gels reduced populations of L. monocytogenes on beef tissue, noncoated tissue became dehy- drated during the 7-day study. Lactic acid treatments alone were only 50% as effective as acid/alginate treatments, whereas acetic acid was more effective than the acetic acid/gel treatment. Numbers of S. Typhimurium were reduced more by exposure to acetic acid/gel treatment than acetic acid alone.

Lactic acid (2%), acetic acid (0.5%), or sodium lactate (4%) alone or in combination with pulse electric field or freezing was ineffective in reducing numbers of E. coli O157:H7 inoculated into beef trimmings (Bolton et al., 2002). Therefore, these treatments were not deemed usable in the control of E. coli O157:H7 in beef burgers.

The combination of acids and heat treatment led to reduction of spore-forming organisms in frankfurters. Frankfurter emulsions incorporated with acids to adjust pH to 5.2 or 4.6 for Bacillus stearothermophilus and pH 4.5 and 4.2 for B. coagulans were heated to 121°C and 105°C or 110°C. Greater inactivation was noted for both organisms at the lower pH level when acetic or lactic acids were used but not with citric, malic, or hydrochloric acid (Lynch and Potter, 1988).

The use of lactic acid for reduction of microbial numbers or pathogens has been similarly applied to pork products. Woolthuis et al. (1984) effectively reduced total plate counts, Enterobac- teriaceae, and Lactobacillaceae counts by 2 to 3 logs, by immersing porcine livers in 0.2% lactic acid for 5 minutes, vacuum packaging, and storing for 5 days at 3°C. A 1% concentration of lactic acid (pH 2.8, 55°C) had little effect on the aerobic plate counts taken from the surface of pork carcasses. Salmonella species or Listeria species were not recovered nor were sensory character- istics affected (Prasai et al., 1992). By increasing the concentration of lactic acid to 2%, numbers of Salmonella species and Campylobacter species were reduced immediately and remained lower 24 hours after slaughter (Epling et al., 1993). Van Netten and Hust In’t Veld (1994) developed a pork skin model system to determine the effect of lactic acid decontamination on microbial counts and changes in predominance of the microflora. Five treatments were delivered to the pork skins: water-treated (control) or exposure at 21°C for 120, 180, 240, or 360 seconds to 2% lactic acid. Not only did the microbial counts decrease as the time of decontamination increased, but the population of microorganisms shifted. Lactic acid was effective in decreasing the number of Enterobacteriaceae as well as other Gram-negative mesophilic and psychrotrophic spoilage organ- isms. Elimination of these groups shifted the population to Gram-positive bacteria and yeasts. Psychrotrophic, Gram-negative bacteria were the most sensitive to lactic acid, followed by mesophilic Enterobacteriaceae, psychrotrophic Gram-positive bacteria; lactobacilli and yeasts were the least sensitive.

The effects of 3% lactic acid at 55°C were studied using pork fat and lean tissue artificially inoculated with 104 _{to 10}5 _CFU/cm2 _{of L. monocytogenes, Y. enterocolitica, Aeromonas hydrophila,}

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P. fragi, or Brochothrix thermosphacta (Greer and Dilts, 1995). After inoculation, tissue was dipped for 15 seconds in water or lactic acid and held for 15 days at 4°C. The spoilage organisms, P. fragi and B. thermosphacta, grew on both tissue types after acid treatment, although tissue treated with acid affected the growth rate. None of the pathogens grew on lean tissue, and A. hydrophila numbers declined by 5 logs within 11 days of storage. Acid treatments of fat tissues led to the reduction of pathogen numbers to nondetectable numbers within 4 days.

Lactic acid dips have also been used successfully in the poultry industry. Total microbial numbers from skin of birds immersed for 15 seconds at 19°C in 1% or 2% lactic acid at pH 2.2 decreased from 5.2 to 3.7 log CFU/g (van der Marel et al., 1988). Levels of psychrotrophs decreased from 3.9 to 2.7 log CFU/g and Enterobacteriaceae from 3.3 to 2.6 log CFU/g. Higher concentrations of the acid did not ensure greater decontamination nor did repeated treatments. A 10% lactic acid and sodium lactate-buffered acid spray (pH 3.0) for chicken legs increased the shelf life from 6 to 12 days at 6°C, and a 2% lactic acid dip at pH 2.3 prolonged shelf life to 8 days (Zeitoun and Debevere, 1990). These treatments inhibited hydrogen sulfide-producing bacteria, such as Pseudomonas species, that contributed to spoilage. The treatments did not affect sensory quality.

Lactic acid (1%) added to both chill water (0°C to 1.1°C, pH 2.8) and scald water (54°C/2 minutes) reduced the bacterial level of broilers inoculated with S. Typhimurium to almost nonde- tectable numbers. Lactic acid added to scald water alone had minimal effect on reducing the numbers of contaminated birds. The number of Salmonella-positive birds was also reduced as a function of time of the dip (Izat et al., 1990a). Lactic acid added to broiler chill water resulted in the develop- ment of a brown coloration. In an effort to eliminate carcass discoloration, reduced levels of lactic acid (0.25%, pH 2.88 or 0.5%, pH 2.62) were combined with propylene glycol (20%) in chill water. Salmonellae were eliminated from broiler carcasses after a 1-hour exposure; however, discoloration still occurred and propylene glycol contributed an objectionable flavor (Izat et al., 1990b). Chicken skin subjected to washing treatments using 1% lactic acid led to significant reductions of Salmonella species and L. monocytogenes compared with monosodium phosphate, sodium tripolyphosphate, sodium acid pyrophosphate, or sodium hexametaphosphate. Consumer evaluation of chicken treated with a 0.5% lactic acid/0.5% sodium benzoate dip concluded that sensory qualities were acceptable (Hathcox et al., 1995; Hwang, and Beuchat, 1995). Pretreatment of broiler carcasses with lactic acid buffer increased shelf life by 6 to 7 days at 4°C and 5 to 6 days at 7°C. When treated broilers were packaged using modified atmospheres, shelf life was lengthened to >36 and 35 days at 4°C and 7°C, respectively (Sawaya et al., 1995).

Lactic acid was more effective than acetic acid when used as a 10-minute rinse for fresh-cut vegetables. Although L. monocytogenes counts were reduced by 0.5 and 0.2 logs, respectively, other compounds, such as chlorine or chlorine dioxide, produced similar reductions of approximately 1 log (Zhang and Farber, 1996).

The interaction of sodium chloride (0.5% to 4%) and pH (4.2) was examined for E. coli O157:H7 grown in TSB at 37°C (Casey and Condon, 2002). E. coli O157:H7 rapidly died at pH 4.2 in media containing lactic, acetic, or formic acids. E. coli demonstrated a 10-fold decrease in numbers when grown in media containing sodium chloride, acidified with lactic acid; however, in sodium chloride- free, acidified media, E. coli decreased 10,000-fold. Sodium chloride offered a degree of protection against the inhibitory activity of lactic acid when the culture was incubated in the medium containing both ingredients, but no protective effect was observed if the sodium chloride was added to the medium 45 minutes after the inoculation. This protective effect was even more pronounced with acetic acid (100,000-fold). The sodium chloride-sparing effect was partially attributed to a reduction in water activity causing the cell size to shrink as a result of loss of water in the cytoplasm and a concomitant increase of internal pH from 5.23 in cells exposed to acid to 5.79 in acid-free media. S. aureus grown at 37°C or 46°C in the presence of 1 M sodium chloride had marked differences in its sensitivity to acetic and lactic acids (El-Banna and Hurst, 1983). Cells grown at 46°C were more heat resistant and better able to grow in media adjusted with acetic and lactic acids to pH 5.0 than cells grown at 37°C.

El-Gazzar et al. (1987) found that A. parasiticus NRRL 2999 grown in a laboratory medium containing 0.5% and 0.75% lactic acid adjusted to pH levels of 3.5 or 4.5 using HCl or NaOH and incubated for 10 days at 28°C produced more aflatoxin B1 than other cultures at 3 days of incubation.

With increasing levels of lactic acid, cultures decreased production of aflatoxin G1. Luchese and

Harrigan (1990) also noted an increase in aflatoxin production adjusted to pH 4.2 using HCl or lactic acid stimulated by substituting half of the carbon content by lactate and a reduction in production at pH 6.8. Aflatoxin production also increased during growth of A. parasiticus in association with L. lactis. A predictive model for growth of E. coli in response to temperature, water activity, pH, and lactic acid concentration has been developed (Mellefont et al., 2003; Presser et al., 1998; Ross et al., 2003).

Toxicology

The LD50 of lactic acid for test animals varied according to animal species (Table 4.2). Infants died

after consuming milk acidified with an unknown quantity of lactic acid. The autopsy revealed hemorrhaging and gangrenous gastritis (Young and Smith, 1944). In two other instances, infants suffered stricture of the esophagus following ingestion of milk acidified with 1 teaspoon of 85% (Pitkin, 1935) or 87.5% lactic acid (Trainer et al., 1945). Premature infants fed acidified milk containing the isomeric forms of lactic acid, D (-) or DL, developed acidosis, weight loss, dehydration,

and vomiting. Therefore, Ballabriga et al. (1970) recommended that the L (+) form should be used

in feeding premature infants. The Food and Agriculture Organization of the United Nations (Food and Agriculture Organization, 1973) supported this view. Hamsters fed a cariogenic diet incorpo- rating lactic acid in the drinking water (40 mg/100 ml) or in the feed (45.6 mg/100 g) did not differ in growth rate compared with control groups (Granados et al., 1949). Some enamel decalcification was apparent in the experimental groups.

Application and Regulatory Status

Lactic acid is a hygroscopic, syrupy liquid having a moderately strong acid taste. Lactic acid is used in the manufacture of jams, jellies, sherbets, confectionery products, and beverages. It is used to adjust acidity in brines for pickles and olives. Calcium lactate is used as a firming agent for apple slices, to prevent discoloration in fruit, and in baking powders. Lactic acid is approved as a GRAS substance for miscellaneous or general-purpose usage (21 CFR 184.1061). The acceptable daily intake for humans is listed in Table 4.3.

LACTATES

Antimicrobial Properties

The salt form of lactic acid has been used successfully as an antimicrobial agent because it provides a slight salty taste that enhances meat flavor, retains color, contributes to water-holding capacity, improves juiciness, increases meat yields, appears naturally in meat products, and extends the shelf life of products. For an excellent review of the antimicrobial nature of sodium lactate, see Shelef (1994) and De Wit and Rombouts (1990).

The MIC of sodium lactate was investigated under optimum growth conditions (pH 6.5, 20°C) for a number of bacteria and yeasts isolated from spoiled and unspoiled meat products (Houtsma et al., 1993). MIC values for Salmonella species ranged from 714 to 982 mM and a narrower range for L. monocytogenes and L. innocua from 804 to 982 mM. Gram-negative bacteria including Pseudomonas and Yersinia had a comparable range to Salmonella, although Campylobacter was particularly sensitive with an MIC of 179 mM. Gram-positive bacteria including lactic acid bacteria, Carnobacterium, Lactococcus, Brochothrix, Bacillus, and Staphylococcus ranged from 268 for S. aureus and Lactobacillus coryniformis to 804 mM for Brochothrix. Yeasts were very broad in

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their sensitivity, with Debaryomyces and Candida being the most resistant at 1161 and 1339 mM, respectively, which might explain their prevalence in spoiled meat products.

The effect of sodium lactate on toxin production, spore germination, and heat resistance was tested on proteolytic C. botulinum strains in peptone yeast extract medium (Houtsma et al., 1994a). Toxin production occurred within 14 days of incubation at 15°C for cultures grown in 0% and 1% sodium lactate but was delayed in the presence of 2% sodium lactate until day 21; no toxin formation was detected after 49 days of incubation in media containing 3% sodium lactate. At a higher temperature of 20°C, toxin formation was noted within 5 days for 0%, 11 days for 1.5%, and 15 days for 2.5%; no growth or toxin formation was detected after 32 days of incubation for media containing 4.0% sodium lactate. At 30°C, toxin production was detected in media containing 4% sodium lactate within 11 days; however, spore germination did not lead to growth after 7 days. Sodium chloride at the same water activity as sodium lactate (0.982) did not inhibit toxin production, indicating that inhibition was not the result of a lowering of water activity.

Lactic acid/sodium lactate and acetic acid/SA were studied in a BHI broth at pH 7, 6, 5, and 4 using 0.1, 0.5, 1.0, and 2 M concentrations (Buchanan et al., 1993). Survivor curves were developed using L. monocytogenes as the target organisms. As expected, the rate of inactivation was dependent on the acid, its concentration, and the resultant pH. Lactic acid was more effective