Effects of Lactates: A Review

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1 Jou17Ul1of Food Protection, Vol. 57, No.5, Pages Copyright, International Association 01 Milk, Food and Environmental Sanitarians Antimicrobial Effects of Lactates: A Review LEORA A. SHELEF Department of Nutrition and Food Science, Wayne State University, Detroit Michigan (Received October 13, 1993/Accepted January 10, 1994) ABSTRACT Sodium lactate is used as humectant and flavor enhancer in meat and poultry products, and there is growing evidence of antimicrobial properties of the salt. Potassium and calcium lactate are equally effective in controlling growth of aerobes and anaerobes in meats, and antibotulinal and antilisterial activities of the lactate anion have been established. The specific action of lactate on the microbial cell is not well understood. No intracellular ph lowering effect could be demonstrated, and the reported small decreases in water activity appear insufficient to explain the effect. Other explanations have been proposed but not yet confirmed. Although lactates appear to be bacteriostatic, their ability to control spoilage and pathogenic bacteria in fresh and processed meat favors their use, particularly in refrigerated meat products in combination with other microbial inhibitors. Key Words: Antimicrobial, lactates, water activity ( )' meat Lactic acid is one of the most widely distributed acids in nature, and together with acetic acid is the most widely employed as preservative. The food-grade acid is produced by controlled fermentation of refined sucrose or other carbohydrate sources. The compound is purified by conversion to crystalline calcium lactate, which is then treated with sulfuric acid to give solutions of the pure acid (24). The acid is also manufactured synthetically by hydrolysis of lactonitrile. Major properties of lactic acid, which is a GRAS additive (3), are summarized in Table 1, and its major uses in foods are summarized in Table 2. Most applications of lactic acid, used for improving the quality of a variety of foods and for controlling microbial growth, are associated with the ph lowering effect. For example, the ph of a 1% aqueous solution of the acid is 2.28, and sprays of 1 to 3% solutions are most often used to sanitize meat surfaces. The treatment results in reduction in viable counts during storage of the carcasses, and is especially effective if done early, before bacteria are frrmly attached to the meat surfaces, since attached organisms tend to be more acid resistant. The decontaminating properties of the acid in meat have been reviewed extensively (10,15,53,60). The psychrotrophic gram-negative bacteria that cause meat spoilage are generally sensitive to the treatment, and a shift towards a predominantly gram-positive flora is favored, For example, decontamination of veal tongues with 2% lactic acid, followed by vacuum-packing, TABLE 1. Properties of lactic acid. A derivative of fatty acid (propionic). M.W Colorless, nonvolatile, viscous liquid (7 cp for 65% solution at 25 C), with acrid taste. GRAS additive for general purposes. Dissociation constant, pka = Very soluble in water. Density (60% solution) = The acid undergoes self-esterification and lactoyllactic acid, CH 3 CH(OH)COOCH(CH 3 )COOH, forms: 4.2% in a 6.3% solution; 12.3% in 55% solution and 33.8% in 88.6% solution. ph of aqueous solutions at 25 C: 1% ; 10% Corrosive; one of the most difficult acids to handle. TABLE 2. Major uses of lactic acid. Cheese curd - adjusting to ph before processing improves flavor, texture and stability. Unsalted butter - improves shelf stability. Egg whites - adjusting the ph before drying improves protein dispersion, powder stability and whipping properties. Egg yolks - improves the shelf stability of mayonnaise. Beer - prevents butyric acid bacteria during fermentation. Bread dough - prevents "ropiness" without affecting yeast fermentation. Hard candy - prevents crystallization (in combination with the sodium salt for buffering effect). Infant foods containing dried milk - precipitates the proteins and renders them more digestible. The acid (0.3%) or its salts act as antibacterials and regulators of intestinal flora. Olives - maintains clarity of the brine. Pickles and relishes - improves taste. Cattle, sheep and poultry carcasses - lowers the viable counts during storage by dipping in solution or spraying (1-3% acid) before bacteria become attached to the surface.

2 increased the shelf-life and decreased the bacterial counts by almost 3 loglo cycles (58). Lowering of the ph by the acid may adversely affect taste and other properties of the food. For example, addition of the acid to fresh meat causes oxidation of myoglobin and darkening of the tissue (10). SALTS OF LACTIC ACID The sodium, potassium and calcium salts of lactic acid are approved for use in foods as direct food ingredients (3). Major properties and uses of these salts are summarized in Table 3. Sodium and potassium lactate (CH)CHOHCOONa, mol wt , and CH)CHOHCOOK, mol wt , respectively) are generally available as 60% aqueous solutions with a neutral ph. They are used as humectants and flavor enhancers in meat and poultry products and contribute to increased cooking yields and water holding capacity (16,45). Levels of 2% (3.3% of the 60% solutions) are used in hot dogs, frankfurters and similar products. Injection of beef roasts with 4% solution of sodium lactate before cooking was reported to increase the cooking yield up to 15%, improve palatability and lower the aerobic plate counts during refrigerated storage (20). Calcium lactate (Ca[CH)CHOHCOO]2' mol wt ) is a hygroscopic powder available in the monohydrate or the more stable pentahydrate form. In addition to its use for dietary calcium supplementation, major applications are in gel formation with low methylated pectin and prevention of "ropiness" in bread dough, as well as a firming agent (Table 3). TABLE 3. Properties of salts of lactic acid. Sodium and potassium lactate Available primarily as 60% solutions with neutral ph. Used as emulsifiers, humectants and ph control agents. (2-3% recommended. ) Flavor enhancers (adjuvants) in meat and poultry products; increase water holding capacity and cooking yields. 2% lactate, based on final weight of the product, is recommended in meat and poultry products. Calcium lactate Available as dry powder, mono- or pentahydrate. Used for calcium supplementation. Firming agent and coloration inhibitor in apple slices. Prevents ropiness (0.2%) and coliform (0.3%) in bread dough and other baked products. Cattle feed additive to regulate the microflora (I % in silage liquor). Used in gel production from demethylated pectins. Improves quality of dried milk powder. LACTATES AS ANTIMICROBIAL ADDITIVES Until recently, there have been scant reports on antimicrobial activities of salts of lactic acid. Growth suppression of Bacillus cereus, B. subtilis and B. circulans was observed in broth containing 0.3% sodium lactate and complete inhibition required 12% of the salt (2). In 1972 Krol (34) reported decreased growth of lactobacilli, micrococci and "Achromobacter" species in dry-cured, country-style ham formulated with sodium lactate. L-lactate at a concentration found in muscle of low ph (ca. 100 mm) prevented anaerobic growth of gram-negative fermentative bacteria (Serratia liquefaciens, Yersinia enterocolitica, Enterobacter cloacae and Aeromonas hydrophila) in buffered broth at ph 5.55 and also prevented aerobic growth of the aeromonad, but no inhibition was seen at ph 6.1 (26). Since 1989, the potential benefits of sodium lactate as an antimicrobial agent spurred interest in research on its effects in meat products. Studies focused on changes in total aerobes and anaerobes in fresh or cooked meats and poultry under various packaging conditions, and specific effects on Clostridium botulinum and Listeria monocytogenes were also reported. A summary of the reports outlining the tested products and lactate levels, storage conditions and the antimicrobial effects is shown in Table 4. Although the sodium salt has been used most often, potassium lactate demonstrated similar antimicrobial effects. The effects at the recommended levels of 2% based on the final weight of the product or even twice that amount were generally bacteriostatic and concentration-dependent. Since in most studies the meats were formulated to simulate commercial products, the effect of lactate was not differentiated from that of its combination with sodium chloride (NaCl), and in some tests also with nitrites. Combinations with NaCl were reported to reduce the effective dose of the lactate salt (1,9,38,50). In addition, pumping brine containing 10% NaCl and 20% sodium lactate into beef roasts showed protection against survival and growth of C. sporogenes and L. monocytogenes in temperature-abused cooked-inthe-bag products (56). MECHANISM OF INHIBITION Studies on the specific action of lactate on the microbial cell are limited (32), but at least two possible mechanisms have been proposed: (i) ability of weak lipophilic acids (e.g., lactic acid) to pass across the cell membrane in their undissociated form, dissociate within the cell and acidify the cell interior (22,29,47); and (ii) specific ability of sodium lactate to lower the water activity (a ) w (13,36). Lowering of intracellular ph In regard to entry of the acid into the cell membrane, the lipophilic acid molecules are able to diffuse freely across the cell membrane in their protonated form (25). Energy-linked carriers and the membrane potential may also be involved in the uptake (33). If the extracellular ph is lower than the intracellular ph, the acid dissociates and releases protons that acidify the cytoplasm. The cell reacts generally to maintain a constant internal ph by removing

3 TABLE 4. Recent reported antimicrobial effects of lactates in meat products. Product Storage d, C Sodium lactate, % Effect Vacuum packed pork liver pate Comminuted cooked turkey, vacuum packed Fresh pork sausage Frankfurters Cooked chicken roll (uncured) Cooked, vacuum packed beef roasts Fresh pork sausage Low fat beef patties Communited cooked beef, 55% m.c. Pork liver sausage, 2% NaCl Fresh pork sausage (40 and 8% fat) Cooked turkey and chicken roll (uncured) 42,6 10,27 45, 5 56,4 49,4 80,0 28,4 12, 5-7 7,20 50,5 12, , " 4 3 b 2" Inhibition of aerobes and anaerobes (13) Delayed toxigenesis in proteolytic C. botulinum (7-8 d) (37) Reduced aerobes; 2 wk extended shelf-life (35) Inhibition of L. monocytogenes and aerobes (4%) (4) Inhibition of L. monocytogenes and aerobes (4%) (4) Reduced aerobes by 3-4% lactate (44) Delayed growth of aerobes (8) Reduced aerobes (17) Inhibition of L. monocytogenes (9) Inhibition of L. monocytogenes (59) Reduced aerobes, psychrotrophs and colifonns (7) Delayed toxigenesis in non-proteolytic C. botulinum (38) " Potassium lactate. b Sodium, potassium or calcium lactate. the protons, and since much of the cell energy is expended to maintain internal ph constant, growth rate is reduced (25). Interference with the proton gradient across the membrane further disrupts cell functions, such as amino acid transport (18,22). This mechanism is supported by observations of increased antimicrobial activity at reduced ph than at near-neutral values and of organic acids (considered nonelectrolytes because of their relatively low ionization constants) being more effective inhibitors than inorganic acids (12,21,30,54). There are indications that lactic acid does not act on the microbial cell in the same manner as do other fermentation acids such as acetic and propionic, and does not behave as predicted on the basis of dissociation constants (40). Although L. monocytogenes is sensitive to lactic acid and its salts, studies on changes in intracellular ph of the organism in the presence of lactic and other weak organic acids showed that the cells maintained a ph gradient of about 1.0 to 1.5 ph unit over a medium ph of 3.5 to 6.5. In response to extracellular lactic acid, the intracellular ph remained near 5.0 even when the external ph was reduced to 3.5 (30). These observations and those of Young and Foegeding (61) suggest that inhibition of L. monocytogenes is not associated with decrease in intracellular ph. Salts of organic acids, such as sodium and potassium lactate, are fully dissociated in aqueous solutions, and at the ph of an unfermented meat product, which is typically , the concentration of the undissociated form of the added lactate is low (5,59) Growth inhibition of bacteria has been attributed to both the proton and the anion (19). Regarding lactate salts, antibotulinal and antilisterial activities of the lactate anion have been established in studies that compared activities of the sodium and potassium salts (9,37). Studies with calcium lactate further supported the antimicrobial activity of the anion (59). Russell (46) suggested that anion accumulation is responsible for the toxic effect of fermentation acids at low ph, but the relationship between ApH across the cell membrane, anion accumulation and acid toxicity has received little attention. No studies appear to have been done on the effect of the lactate anion at near neutral ph. Effect on water activity There is some evidence that bacterial inhibition by salts of lactic acid may be associated with effects on water activity (a )' w When we first investigated antilisterial effects of sodium lactate, initial tests were conducted in broth (50). Results in the liquid medium showed that only at high concentrations of sodium lactate, exceeding 5%, was growth suppressed. Although on an equal concentration basis antimicrobial agents have been generally less effective in food than in broth, we found that lower listeriostatic lactate concentrations were required in cooked meat and poultry than in broth, and they decreased as moisture, values, and temperature were reduced (9,50). Other studies using broth as the growth medium also confirmed limited or no microbial inhibition by lactate (e.g., 41), whereas addition to meat products generally demonstrated antimicrobial effects, as evident from data in Table 4. The effects of lactates on water activity were studied in aqueous solutions and in meat products. Chirife and Ferro Fontan (11) studied the effect of sodium lactate in aqueous solutions and observed no exceptional lowering properties. Indeed, they found that at equal concentrations lactate was inferior to NaCI in lowering the a ' w in contrast to previous reports (36). De Wit and Rombouts (14) compared growth of several spoilage and pathogenic organisms in broth in the presence of sodium lactate or NaCI at an equal water activity of The sodium concentration in the medias also the same. With the exception of a strain of Escherichia coli, all test organisms displayed sensitivity to lactate but not to chloride. The authors concluded that the antibacterial effect of sodium lactate could not be ascribed to the water activity lowering effect (14). As to measurements in meat products, Hammer and Wirth (27) reported values in liver sausage mixes after additions of I% of various sodium salts including: acetate, ascorbate, chloride, citrate, diphosphate, glutamate and lactate. Sodium chloride was most effective in reducing the a ' w followed by lactate, which produced 66% of the reduction by the chloride. Measurements of changes in both bacterial growth and in meat products by the addition of sodium lactate have been reported by a number of researchers. A

4 summary of these data is presented in Table 5. Either sodium lactate or a combination of sodium lactate and chloride were employed in these studies. The of the lactate-free products ranged from to (mean = 0.970). Although different products, microorganisms, levels of lactate and chloride, and methods for determination were used, in each of the studies the level of lactate that inhibited bacterial growth in meat also lowered the a ' w even though the change was small. The critical range was to (mean = 0.970) and the mean difference was (Table 5). In our studies on effects of lactates on growth of L. monocytogenes in a meat model system consisting of an additive-free comminuted beef, the addition of 4% sodium lactate to meat with 55% moisture completely inhibited growth of strain Scott A when held at 20 C for 7 days (9). Measurements of showed that NaCl was more effective than sodium lactate in lowering the of the meat (Table 6), in agreement with findings of Chirife and Ferro Fontan (11), but 4% NaCl alone did not inhibit growth whereas 4% sodium lactate did. Resistance of L. monocytogenes to NaCl is well documented despite its lowering effect. It may be concluded from the data summarized in Tables 5 and 6 that although sodium lactate lowers the in meat, the effect is small and insufficient to fully explain the antimicrobial effects. The specific data on inhibition of L. monocytogenes by lactate and the corresponding measured can be compared further to other published studies on effect of on growth of the organism. Several factors affect the ability of the organism to survive at low a ' w including temperature, salt combinations, and ph, as seen from the summary of the studies (Table 7). For example, Miller (39) studied growth and survival of L. monocytogenes Scott A in brain heart infusion (BHI) broth at various levels using either NaCl, glycerol or propylene glycol as humectants. The corresponding minimal levels for growth at 28 C were 0.92, 0.90 and 0.97, respectively. Nolan et al. (43) used either NaCl, sucrose or glycerol in tryptic soy broth-yeast extract (TSB- YE) to determine minimal levels for growth during incubation at 21 C for up to 21 d. Minimal levels were 0.92, 0.92 and 0.90, respectively, and the presence of 0.6% of yeast extract in TSB increased the tolerance of L. monocytogenes to all three humectants when compared to TSB alone. Despite the variations in testing conditions and humectants, the values for the minimum for growth of listeriae with NaCl as humectant range from 0.90 to 0.94, with a mean (n = 5) of (Table 7). Overall, these values are lower than those reported in meat products containing lactate (mean = 0.954). If a critical is assumed to be the same in broth and in food, then the inhibition of listerial growth observed in lactate-treated meats at a higher can be attributed to an action of lactate that is unrelated to a. The observations of listerial control w at a higher than predicted support the efficacy of a barrier system, whereby bacteriostatic effects in foods can be attained at a higher by a combination of additives. Water activity has become a universally accepted, practical measurement to predict shelf-stability and microbiological safety of foods, but questions and debates on its value have been raised in recent years. Slade and Levine (52) proposed that polymeric solutes may produce more stable systems with greater microbiological stability than non polymeric small compounds such as sugars. Since lactic acid undergoes self-esterification and polymerization in aqueous solutions (28; see also Table 1), such changes may have an effect on the availability of water for microbial growth. Other effects Other explanations have been proposed for the antimicrobial effect of lactate. It was suggested that in the inhibition of C. botulinum high levels of lactate ions may shift the reduction of pyruvate to lactate closer to its thermodynamic equilibrium and thereby inhibit a major anaerobic energy metabolism pathway essential for growth (37). Still another plausible explanation proposed by these authors for C. botulinum was that lactate efflux from the bacterial cell may be coupled to adenosine triphosphate (ATP) generation from proton transfer across cell membranes. This may be inhibited by a high level of extracellular lactate, as demonstrated in membrane vesicles from Streptococcus faecalis (51). Hydroxycarboxylic acids (citric, lactic, malic and tartaric) are known for their chelating properties. Together with citrates, pyrophosphates and ethylenediaminetetraacetic acid (EDT A), lactates are the most commonly used chelators in foods (31). For example, the stability constant of Fe (III) for lactic acid is 6.4 (23). This value can not predict the formation efficiency of an iron-lactate complex in a food system such as meat, which contains numerous interfering constituents, and is lower than the value for citrate (11.85). However, chelation of iron in meat may contribute to the antilisterial activity of lactate. This is supported by observation that lactate stabilizes fats and oils, probably by chelating trace amounts of iron (42). Moreover, we have observed enhanced antilisterial activity of lactates in meats TABLE 5. Effects of sodium lactate on water activity and microbial growth in meat products. Product Storage dloc NaUNaCl % Product (no lactate) Critical Organisms Reference Pork liver pate Cooked ham Comminuted, cooked beef Pork liver sausage 42/6 60/5 7/20 50/5 2/2 1/2.2 4/0 4/2 2/4 3/ Total aero anaer. Total aerobes L. monocytogenes L. monocytogenes (13) (57) (9) (59)

5 Salt Sodium chloride Sodium lactate " Adapted from (9). "a of control was w Water acti vity ( )" Salt concentration, % TABLE 7. Minimal water activity levels for growth and survival of L. monocytogenes in humectant-broth systems. Humectant Testing Minimum Reference conditions 16% NaCI I yr (48) 10% NaCl (6) 10.5% NaCI 15 d, 37 C <0.80 (49) 20-30% NaCI 5 d, 37 C (49) 30.5% NaCI 100 d, 4 C (49) NaCI 20 d, 4 C 0.93 & 0.94" (55) Sucrose 0.92 & 0.94" (55) Glycerol 0.91 (55) NaCI 20 d, 30 C 0.90 (55) Sucrose 0.91 & 0.94" (55) Glycerol 0.89 (55) NaCl d, 28 C 0.92 (39) Glycerol 0.90 (39) Propylene glycol 0.97 (39) NaCI 21 d, 21 C 0.92 (43) Sucrose 0.92 (43) Glycerol 0.90 (43) " For initial populations of IQ4and 10 2 CFU/ml, respectively. b For strain Scott A and Brie I, respectively. following heat treatment (59). A comparison of the antilisterial effects of sodium lactate in pork liver sausage showed superior growth inhibition in sausage that was sterilized (15 min, 121 C) after addition of the lactate than in a heat processed product (water bath, to meat internal temperature of 70 C). Processing conditions in the former may have contributed to increased chelation of polyvalent cations that are essential for listerial growth. It is possible that the effects of lactate on C. botulinum are also associated with chelation of polyvalent cations such as iron. The observations of enhanced antilisterial activity of lactate in meats as compared to broth, and increased activity with a decrease in moisture content, suggest the possibility that bacterial growth inhibition is determined by the concentration of lactate in the water phase. Since growth of L monocytogenes is inhibited by 4% sodium lactate in meat with 55% moisture, the concentration in the water phase of the meat is >7% if all, or most, of the lactate is in the water phase (9). This explanation reconciles the discrepancies in the inhibitory concentrations of lactate in broth and meat. There is now sufficient evidence for antibacterial effects of the sodium, potassium and calcium salts of lactic acid in fresh and processed meat products. In addition to growth of aerobes and anaerobes that is controlled in the presence of lactates, at least two pathogens, C. botulinum (both proteolytic and non-proteolytic strains) and L monocytogenes, are also affected. Hence, there are benefits of added safety for meat products containing lactates, particularly during extended shelf-life at refrigeration temperatures. A number of explanations have been considered for the antimicrobial effect of the salts at neutral or near neutral ph, including acidification of the cell cytoplasm and lowering of a. No one single reason appears satisfactory at this time in e~plaining the effects. Observation of more pronounced antibacterial effects in meats than in broth emphasizes the need for further studies of the activity of lactates in food systems. REFERENCES 1. Anders, R. J., J. G. Cervany and A. L. Milkowski Method for delaying Clostridium botulinum growth in fish and poultry. U.S. Pat. 4,798,729 Jan. 17 and 4,888,191 Dec Angersbach, H Systemic microbiological and technological investigation into improving the quality of foods of animal origin. vol. III. Influencing the growth of three Bacillus types by means of sodium chloride, sodium acetate, sodium diacetate, sodium citrate, sodium lactate and sodium tartrate. 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