Growth Suppression of Listeria monocytogenes by Lactates in Broth, Chicken, and Beef

Transcription

1 283 Journal of Food Protection, Vol. 54, No. 4, Pages (April 1991) Copyright International Association of Milk, Food and Environmental Sanitarians Growth Suppression of Listeria monocytogenes by Lactates in Broth, Chicken, and Beef LEORA A. SHELEF* and QIAN YANG Department of Nutrition and Food Science, Wayne State University, Detroit, Michigan (Received for publication August 20,1990) ABSTRACT Sodium or potassium lactate is available commercially as a neutral aqueous solution (60%), approved for use as a flavoring agent in meat and poultry products. While recommended also for extending shelf life, little work on its antimicrobial effects has been published and none in relation to Listeria monocytogenes. Studies in tryptic soy broth showed that concentrations higher than 5% delayed growth of three strains of L. monocytogenes. Experiments in sterile comminuted chicken and beef at 35, 20, and 5 showed growth suppression by 4% lactate, which increased with decrease in storage temperature. The organism was consistently more sensitive to lactate in beef than in chicken, displaying an extended lag phase of 1-2 weeks at 5 C. Combinations of lactate (4%) with NaCl (3%) or nitrate (140 ppm) did not enhance the effect. Lactate did not alter the beef or chicken ph, and no difference was observed between the effect of the two salts, inferring that the lactate ion is responsible for the delay in listerial growth. Sodium lactate is known for its humectant activities and as a ph control agent. Its use as an additive in baked products at levels of 1.5-2% was reported as early as 1969 (15). The salt is also approved by the USDA as a flavoring agent in meat and poultry products at a level of 2% based on its solid weight. In addition to the meat flavor enhancement, the humectant properties contribute to the waterholding capacity and increase the cooking yields. Applications recommended by manufacturers of lactates include addition of 2-3%, based on the finished product weight, to fresh sausage and a variety of cooked meat and poultry products (8). The antimicrobial activity of lactic acid, from which the salt is derived, is well documented and is associated with reduction of ph to levels below the growth of a large number of bacteria. Antimicrobial effects are attributed also to lactate, as evidenced by product information supplied by manufacturers (4). However, published studies on the antimicrobial activity of lactate salts in meat or other foods are limited. Angersbach (2) reported that sodium lactate at concentrations of up to 7% in meat resulted in complete inhibition of Bacillus spp., including B. cereus, B. subtilis, and B. circulans. Decreased growth of bacterial species was observed in dry cured ham formulated with sodium lactate (12). Lactobacilli, micrococci, and "Achromobacter" were affected. Debevere (6,7) studied effects of lactate on water activity and shelf life of refrigerated vacuum-packaged pork liver pate. Addition of 2% sodium lactate decreased the water activity and inhibited growth of lactic acid bacteria that are normally responsible for spoilage of the product. Growth of Clostridium botulinum was delayed in fish, chicken, and turkey treated with 1.5 to 3.5% sodium lactate (/). In addition, delayed C. botulinum toxin production by sodium lactate was reported in turkey products (13). In the latter study, vacuum-packaged comminuted raw turkey that contained 2-3.5% sodium lactate showed delayed toxin formation after inoculation with C. botulinum, and the antibotulinal effect was concentration-dependent. Effects in vacuum-packaged beef roasts injected with a solution of sodium chloride, tripolyphosphate, and various levels of lactate before cooking were reported recently (9,14). Data showed delayed microbial growth with increasing levels of sodium lactate to 4% during storage at 0 C, while a concentration of up to 3% was judged optimal for overall beneficial effects, which also included cooking yield, flavor, and color. The microflora after 84 d of storage became more uniform, consisting predominantly of heteroand homofermentative Lactobacillus spp. While it has been suggested that growth inhibition by lactates is affected irrespective of the microbial types (5), currently published data are insufficient to conclude whether lactate is equally effective in controlling spoilage and pathogenic microorganisms, or whether it favors growth of certain organisms over that of others, bringing about flora alteration. Product information by manufacturers refers to proven effective inhibition of Listeria and Salmonella spp. (4), but no studies on the effect of lactate on Listeria monocytogenes (LM) appear to have been published. The limited reports on the antibacterial activity of lactates raised the interest to study their effects on growth of LM. The purpose of the present study was to investigate the antilisterial effects of sodium lactate in laboratory media and in meat model systems. Since elevated sodium levels in meat or poultry may result from the combination of sodium lactate with sodium chloride, the antimicrobial effects of potassium lactate were further examined and compared to those of the sodium salt. In addition, the combined effects of lactate, sodium chloride, and nitrite were investigated.

2 284 SHELEF AND YANG MATERIALS AND METHODS Bacterial cultures LM strains Scott A, associated with the 1983 milk-borne listeriosis outbreak, Brie-1, a Brie cheese isolate, and V-7, originally isolated from milk, were used in the studies in broth, while the former was used in studies in meat. Stock cultures of the strains were maintained on tryptic soy agar (TSA) at 5 C. Inocula for the experiments were prepared in tryptic soy broth tubes (TSB, Difco Labs, Detroit, MI), incubated at 35 C for 24 h. Cultures were appropriately diluted in TSB so that the initial CFU/ml or g was about Studies in broth TSB tubes containing increasing concentrations to 10.5% by weight of sodium or potassium lactate (NaL or KL, 60% aqueous solution, Food Grade, Wilke International, Inc., Overland Park, KS) were prepared and sterilized. After cooling to room temperature, the broth was inoculated with 0.1 ml of the test organisms and incubated at 35 C. Initial cell numbers, and those during incubation, were determined using prepoured TSA plates and a spiral plater (Spiral System, Inc., Bethesda, MD). All sample dilutions were made in 0.1% sterile peptone water and colonies were enumerated after incubation for 48 h at 35 C. All tests were run in duplicate and repeated twice. Studies in meat model systems Foods used in the study were comminuted beef or chicken with added broth (Baby food, H.J. Heinz Co., Pittsburgh, PA). Their composition is shown in Table 1. The effects studied included those of the following ingredients and concentrations, alone or in combination : NaL or KL, 2.6 and 4%; NaCl, 2 and 3%; and KN0 2, 140 ppm. After adding the test compounds to the food, the contents were thoroughly mixed and 11 -g samples were dispensed into 50-ml autoclavable plastic beakers. The beakers were covered and sterilized (121 C, 15 min). After cooling to room temperature, the samples were inoculated with LM strain Scott A. Chicken and beef alone, containing levels of water equal to those added from the aqueous solutions of the lactates, served as control. Incubation was at 5, 20, and 35 C. Cell numbers were determined immediately after sample inoculation and at time intervals during storage. For the determination, samples were diluted 1:10 with peptone water in a sterile stomacher bag and macerated for 2 min using Model 400 Stomach (Dynatech Laboratories, Inc., Alexandria, VA). The suspension (0.1 ml) was either directly spread on prepoured plates or further diluted and plated using the spiral plater. Incubation and enumeration were as described before. Two replicates were tested for each combination of compounds and storage time, and mean values calculated. Determination of ph The ph of representative samples containing the tested concentrations of NaL or KL was determined after sterilization using an Orion ph meter. RESULTS The effects of increasing NaL concentrations on growth of three LM strains are summarized in Table 2. Inocula (log CFU/ml) in these experiments were While cell multiplication occurred even in the presence of the highest NaL concentration, the extent of bacterial growth decreased with increase of the salt content, and all three strains behaved in a similar manner. Partial growth curves for strain Scott A in TSB alone and in combination with NaL are shown in Fig. 1. Cell numbers generally reached a maximum after 24 h of incubation at 35 C and declined after 48 h. Reduced growth after 24 h was seen only by the addition of 7.8% NaL, and these effects persisted throughout the incubation period. TABLE 2. Effects of sodium lactate on cell numbers of Listeria monocytogenes strains. Strain NaL in TSB (% hy weight) Scott A Brie-1 V Log 1() CFU/ml after 24 h at 35 C. Initial counts were: Scott A, 4.2; Brie-1, 3.74; V-7, % NaL 5.2% NaL 7.8% NaL i Incubation at 35 C, h Figure 1. Effect of sodium lactate on growth ofl. monocytogenes strain Scott A in trypticase soy broth at 35 C. 80 TABLE 1. Analysis of the meat products. Component Water Protein Fat Ash Carbohydrate H. J. Heinz, * = No significant amount. Beef % Chicken % Preliminary examination of effects of two lactate concentrations on growth of LM in refrigerated beef confirmed growth suppression. Examination of growth of strain Scott A in meat alone and with added KL during refrigeration for 30 d showed effects with 2.6% of the salt, and these were enhanced by the addition of 4% (data not shown). This latter concentration was used for further studies. Growth curves for strain Scott A in beef and chicken

3 DELAYED GROWTH EFFECTS OF L. MONOCYTOGENES BY LACTATES IN MEAT 285 containing or KL during incubation at 35 C for 68 h are shown in Fig. 2. Maximum populations in the control samples exceeded 10 9 CFU/g. Growth was reduced in all lactate containing samples, but effects were small in chicken and more pronounced in beef. Effects during storage at 20 C for 8 d are shown in Fig. 3. Maximum cell numbers exceeded 10 9 as before, and although growth was very similar in chicken and beef alone, the growth suppression effects were more pronounced in beef. Suppressed growth was seen also during refrigeration at 5 C for 21 d (Fig. 4). While maximum cell numbers in control samples were ca CFU/g, LM grew faster in chicken than in beef. As before, the lactates were less growth reduction at this temperature, but a very small difference was discerned in growth pattern of the organism in control and NaCl containing samples at 20 or 35 C. The effects of KL alone or in combination with NaCl were identical during the first 3 weeks of storage at 5 C, but on further storage the organism grew at a slower rate in the presence of the two salts. Similar results were seen also in the higher incubation temperatures (data not shown). The effects of potassium nitrite (140 ppm) alone, and in combination with lactate, were examined during storage at 20 C (Fig.6) and 5 C (Fig. 7). The presence of nitrite in beef did not alter the growth pattern of LM. Likewise, the combination of nitrite with either NaL or KL (4%) did not enhance the inhibitory effects of the lactates. NaL and KL additions to TSB produced small ph lowering effects. TSB alone had a ph of 7.23, and addition of 10% lactate lowered the broth ph to 6.7. There was no difference in ph of the meats alone and with 4% added lactate; beef ph was 6.27, and chicken ph was Incubation at 35 C, h Inclination at 35* C, h Figure 2. Effect of 4% sodium or potassium lactate on growth of during incubation at 35 C Figure 4. Effect of 4% sodium or potassium lactate on growth of during incubation at 5 C. Incubation at 20 C, days Incuhalion at 20*C, days Figure 3. Effect of 4% sodium or potassium lactate on growth of during incubation at 20 C. effective inhibitors in chicken than in beef, where no growth was visible until the 10th d of storage. Suppressed growth persisted even after 21 d, when populations were 2-4 log cycles lower than in control medium. In contrast, by the end of this refrigeration period, cell numbers in treated chicken reached or approached those in lactate free samples. Since NaCl is generally present in cooked meat products, the effect of 2 and 3% additions to beef was investigated alone and in combination with lactate. The lower salt concentration had negligible effects on growth (data not shown). Growth curves for LM in beef containing 3% NaCl,, and the combination of the two during storage at 5 C are shown in Fig. 5. NaCl caused some Figure 5. Effect of 4% potassium lactate, 3% NaCl, and their combination on growth of L. monocytogenes strain Scott A in beef during storage at 5 C.

4 Incubation at 20 C, days Con 4% 4% 140 Nal KL SHELEF AND YANG trol NaL KL ppm KN0 2 + KNO, + KNO, Figure 6. Effect of 4% sodium or potassium lactate, 140 ppm potassium nitrite, and their combination on growth of L. monocytogenes strain Scott A in beef during incubation at 20 C Incubation time at 5 C, days 140 ppm KN0 2 NaL + KN0 2 KL + KNO, Figure 7. Effect of 4% sodium or potassium lactate, 140 ppm potassium nitrite, and their combinations on growth of L. monocytogenes strain Scott A in beef during storage at 5 C. DISCUSSION The meat products used in this study were selected for preliminary evaluation of antilisterial effects of lactates. They are convenient to use and, being free of additives, they lend themselves to evaluation of effects of meat products ingredients. The inoculum of LM used in the experiments was approximately 10 3 CFU/g, substantially higher than contamination levels reported in fresh or processed meats. Although it is possible that the antilisterial efficiency of lactates will be higher at lower contamination levels, this may not be the case if small numbers of listeriae are found in the presence of other bacteria, particularly if the effect of lactates is nonspecific with regard to genus and species. Comparison between results obtained by the use of the Na or K salts revealed no difference, confirming that the two salts are equal in their effect. Partial replacement of the Na salt with the K salt could be considered in order to limit the Na ions in meat products if growth suppression of other bacterial species is also equally affected by either salt, and product flavor is acceptable. Sodium chloride or potassium nitrite at concentrations permitted in foods did not affect growth of LM, nor did they enhance the effects of lactate. These observations confirm previous published data (//) on resistance of LM to salt and nitrites in meat products. With regard to the mode of action, Hammer and Wirth (10) showed that sodium lactate can lower the water activity in cooked liver sausage, and similar effects were reported also in cooked ham (3). In a study by Debevere (7), using vacuum-packaged coarse pork liver pate with 50% H,0, the water activity of decreased to by the addition of 2% NaL, and counts of lactic acid bacteria after 6 weeks at 36 C were three-log cycles lower than in control. However, similar lowering of the water activity, achieved by additions of NaCl, failed to affect microbial growth. In the present study, growth suppression by the addition of 4% lactate was higher in beef than in broth. This is in contrast to the more pronounced inhibitory effects normally observed in tests conducted in laboratory media, suggesting that restriction of free water may indeed be a contributing factor. Since the comminuted beef and chicken used in the present study contained substantially higher moisture content (75-77%) than most meat products, the effects of lactate may be enhanced in those with lower a w. The effect of a w reduction on bacterial growth is characterized by extended lag phase and suppressed logarithmic growth rate (16), and such growth patterns were observed for LM in lactate containing beef, particularly during refrigerated storage. Growth curves for exterior and interior aerobic plate counts in cooked beef roasts containing during storage for 84 d at 0 C also had similar characteristic shapes (14). It was suggested that a mechanism other than lowering of water activity is responsible for the antibacterial effects (3), and that inhibitory effects result from lactate transport into the bacterial cell. Maas et al. (13) proposed that delay of botulinal toxin production may be caused by a shift in the pyruvate to lactate reaction by the high levels of lactate ions, thereby inhibiting a major anaerobic energy metabolism pathway essential for growth. The effect of high levels of extracellular lactate on Listeria cells is not known at this time. Results indicated growth suppression of LM by the lactates for one week or longer during refrigeration temperatures and higher sensitivity of the organism in beef than in chicken. The fat content is higher in the latter (10.1% vs. 7.5% in beef), and this might have provided protection to the cells. Addition of Na or K lactate to the foods, unlike that of lactic acid, did not influence their ph. Research on the effect of lactate on LM alone and in the presence of other foodborne bacteria is needed in order to determine if the effects are species-specific. Such information will enable to assess the benefits of this compound as an antimicrobial in meat products. The present studies do not confirm manufacturers' claim to the effectiveness of lactate at allowable concentrations in products containing ca. 75% water. Further studies are in progress to determine antilisterial activities of lactates in products with reduced moisture and the influence of lactates on a. JOURNAL OF FOOD PROTECTION. VOL. 54, APRIL 1991

5 DELAYED GROWTH EFFECTS OF L. MONOCYTOGENES BY LACTATES IN MEAT 287 REFERENCES 1. Anders, R.J., J.G. Cerveny, and A.L. Milkowski Method for delaying Clostridium botulinum growth in fish and poultry. U.S. Patents #4,798,729. Jan. 17, and #4,888,191. Dec Angersbach, H Systematic microbiological and technological investigation into improving the quality of foods of animal origin. III. Influencing the growth of three Bacillus types by means of sodium chloride, sodium acetate, sodium diacetate, sodium citrate, sodium lactate, and sodium tartrate. Fleischwirtsch. 51(2): Anonymous A meaty problem solved. Food Processing, December. p Anonymous Application of sodium lactate and potassium lactate in processed meat and poultry. Wilke International, Inc., Overland Park, KA. 5. Bacus, J Microbial control methods in fresh and processed meats. Recip. Meat Conf. Proa, Vol. 41. p Debevere, J.M The use of buffered acidulant systems to improve the microbiological stability of acid foods. Food Microbiol. 4: Debevere, J.M Extended shelf-life from lowered A and growth inhibition of lactic acid bacteria by sodium lactate. Fleischwirtsch. 69: Duxbury, D.D Natural sodium lactate extends shelf life of whole and ground meats. Food Processing, January, p Duxbury, D.D Sodium lactate extends shelf life, improves flavor of cooked beef. Food Processing, April, pp Hammer, G.F., and F. Wirth Diminution of water activity (a w ) in liver sausage. Proc. Eur. Meet. Res. Workers, No. 31, p Junttila, J., J. Him, and E. Nurmi Effect of different levels of nitrite and nitrate on the survival of Listeria monocytogenes during the manufacture of fermented sausage. J. Food Prot. 52: Krol, B Meat products. Voedingsmiddelen-Technologie 3: Mass. M.R., K.A. Glass, and M.P. Doyle Sodium lactate delays toxin production by Clostridium botulinum in cook-in-bag turkey products. Appl. Environ. Microbiol. 55: Papadopoulos, L.S., R.K. Miller, G.R. Acuff, C. Vanderzant, and J.T. Keaton The effect of sodium lactate on cooked beef shelf-life, chemical attributes, and sensory properties. A final report to the Beef Industry Council, National Live Stock and Meat Board. Texas A&M University, College Station. 15. Reid, T.F Lactic acid and lactates in food products. Food Manufacture 44(10): Trailer, J.A Sanitation in food processing. Academic Press, Inc., New York. p. 127.