Protein variations in Listeria monocytogenes exposed to sodium lactate, sodium diacetate, and their combination.

Most studies of the effect of adverse conditions on survival of Listeria monocytogenes have focused on stress caused by acid or sodium chloride. However, no information is available on resistance of this pathogen to stress caused by salts of organic acids. Sodium lactate and sodium diacetate are generally recognized as safe substances and are approved as ingredients for use in foods. We evaluated antilisterial properties of each of these salts and the enhanced inhibition effected by their combination in ready-to-eat meat products at pH 6.3. Changes in proteins found in this pathogen were studied in the presence of the salts in a chemically defined medium at the same pH using a proteomic approach. The total numbers of protein spots obtained from two-dimensional electrophoresis were 198, 150, and 131 for sodium diacetate, sodium lactate, and the control, respectively. Sodium diacetate treatment produced the highest number of unmatched proteins (124 versus 53 in lactate), the greatest increase in expression (20 versus 5 in lactate), and the highest number of novel proteins (90 versus 45 in lactate). The number of repressed proteins was highest in the combination treatment (41 versus -30 in the single salt treatment). Six proteins that increased or decreased by > or = 10-fold were further investigated; oxidoreductase and lipoprotein were upregulated, and DNA-binding protein, alpha amylase, and two SecA proteins were downregulated or completely suppressed by the salt treatment. Identification of all protein spots is essential for comparison with proteins induced or suppressed under other stress conditions.

Factors affecting the antilisterial effects of nisin in milk.

The ability of Listeria monocytogenes to proliferate in milk and the antilisterial activities of nisin are well documented. Although milk fat was reported to reduce the antimicrobial activities of nisin, there is little information on the influence of milk fat on the antilisterial activities of nisin in refrigerated milk, and whether pasteurization and homogenization influence these activities. Fresh, pasteurized, and homogenized milk samples (0.1%, 2.0%, and 3.5% fat) were treated with nisin (0-500 IU/ml) and challenged with 10(4) CFU/ml L. monocytogenes strain Scott A. The organism was most sensitive to nisin in skim milk, showing rapid decline in cell numbers to <10 CFU/ml after 12 days at 5 degrees C following treatment with 250 IU/ml. An initial decline in cell numbers in 2% and whole milk was followed by regrowth of the organism. Loss of the antilisterial effects of nisin was confirmed in homogenized whole milk, whether raw or pasteurized, but not in raw or pasteurized whole milk that was not homogenized. Tween 80, a nonionic emulsifier, partially counteracted the loss of the antilisterial activity of nisin, whereas lecithin, an anionic emulsifier, had no effect. These results demonstrate that the chemical composition and treatment of foods may play an important role in the antilisterial effects of nisin.

Automated measurements of antilisterial activities of lactate and diacetate in ready-to-eat meat.

Standard procedures to enumerate Listeria organisms rely on plating food samples on selective agar media. The procedures are labor-intensive, and because of the limited sensitivity, pre-enrichment step is required for the detection of low numbers of the pathogen. In the present study, an automated rapid optic procedure and the standard procedure were used to determine the behavior of the pathogen in ready-to-eat (RTE) meat and to test the effect of antilisterial agents. Listeria monocytogenes strain Scott A or a six-strain mixture of Listeria was studied using lactate (2.5%), diacetate (0.2%) and their combination in beef bologna and in sterile beef emulsion. Samples stored for up to 60 days at 5 and 10 degrees C were tested at time intervals during storage. Using the plate count method, each of the salts caused a delay in growth of the pathogen, and the salt combination was most effective causing listeriostatic effects and decline in growth of the pathogen at 5 degrees C. High negative correlation (r), ranging from 0.92 to 0.99, was obtained between the detection time (DT) recorded by the optic procedure (BioSys instrument) and cell numbers determined by the plate count procedure. The rapid (< 24 h) optic procedure was reliable in assessing the efficacy of antimicrobials and in rapid detection of low levels of listeriae that are undetectable by direct plating procedure.

Sensitivity of Six Strains of Listeria monocytogenes to Nisin.

The sensitivity of six strains of Listeria monocytogenes (serotype 1, 1a, 2, 4a, 4b, and a nonhemolytic strain) to nisin was compared at pH 5.0, 6.0, and 7.0. The effects of 240 and 600 IU/ml of nisin in broth on 103, 104, 106, and 108 CFU/ml of each of the strains were tested, and cell numbers were determined after incubation for 30 min, 60 min, and 24 h at 35°C. Although populations decreased after a contact time of 30 min, survivors of 240 IU of nisin per ml reached high numbers after 24 h. Similar results were obtained for 600 IU of nisin per ml but there were no survivors after 24 h of incubation when inocula of less than 104 CFU/ml were tested irrespective of the strain. The sensitivity of L. monocytogenes to nisin at each pH was strain dependent, and it was enhanced at the low pH.

Rapid Optical Measurements of Microbial Contamination in Raw Ground Beef and Effects of Citrate and Lactate.

The levels of microbial contamination in purchased pre-wrapped fresh ground beef samples were estimated by using the BioSys™ automated optical system for rapid detection of the presence of microorganisms. Additionally, the response of the system to the activity of antimicrobial agents in meat was evaluated after adding (separately) the sodium salts of lactic and citric acid to meat samples. Meat homogenates in peptone water were incubated at 30°C in broth containing the indicators bromcresol purple (pH) or resazurin (redox potential). Curves (light transmittance versus time) generated by the instrument showed significant and rapid changes in the indicator color, and times of detection of the initial changes were inversely proportional to the standard plate counts in the meat samples. This relationship indicated that the detection time can provide a good estimate of the microbial quality of fresh meat. Treatment of meat with 2% citrate or 2% lactate extended the shelf life by about 2 and 4 days. respectively. Detection time values confirmed the ability of the salts to delay the microbial spoilage of meat.

Survival of Listeria monocytogenes in Cottage Cheese.

Most studies on the behavior of Listeria monocytogenes in cheeses have focused on the soft, ripened types because of human listeriosis outbreaks linked to consumption of these foods, while observations in cottage cheese are limited and conflicting. Fresh market cottage cheese samples with and without sorbate, purchased in the United States and Canada, respectively, were contaminated with L. monocytogenes strain Scott A (103 CFU/g). Mean total plate counts/g, log10, in the fresh U.S. and Canadian cheeses were 3.54 and 5.22, respectively. After 24 days at 5°C, the respective numbers were 3.51 and 6.27. Mean pH values in the U.S. cheeses at freshness and after 24 days were 5.05 and 5.03, respectively, and values in the Canadian cheese were 4.89 and 4.77. Increases of >3 logs in cell numbers were seen at temperature abuse (10 and 20°C) in all cheese samples, although sorbate-containing cheese spoiled at a slower rate and samples were free of visible surface mold growth even after 21 days. Cottage cheese did not support growth of L. monocytogenes at any of the storage temperatures, and declines (0.35 and 0.81 log10, in sorbate-free and sorbate-containing cheese, respectively) were seen after 24 days at 5°C. Treatment with 3% sodium or calcium lactate did not affect listerial cell numbers. These findings demonstrate that sorbates in cottage cheese are effective as preservatives but not as antilisterials. Persistence of listeriae in cottage cheese emphasizes the need to prevent contamination during manufacturing and storage.

Antimicrobial Effects of Lactates: A Review.

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.

Relationship Between Water Activity, Salts of Lactic Acid, and Growth of Listeria monocytogenes in a Meat Model System.

The relationship between water activity (aw), lactate, and growth of Listeria monocytogenes strain Scott A was studied in a meat model system consisting of cooked strained beef ranging in moisture content from 25 to 85% (wt/wt). Lactate (4%) depressed meat aw, and differences between aw values in control and lactate-treated samples at each moisture level increased progressively with decrease in moisture, from 0.003 (85% moisture) to 0.046 (25% moisture). Maximum cell numbers per g in control samples stored at 20°C for 7 d were about 109 (45-85% moisture, aw= 0.981-0.994) and 107 (35% moisture, aw = 0.965); there was no growth in meat with 25% moisture (aw = 0.932). Sodium lactate (4%) suppressed listerial growth at >55% and inhibited growth in samples with 25-55% moisture (a < 0.964). Lactate concentrations less than 4% were not listeristatic, but combinations of 2 or 3% lactate with 2% NaCl in samples with 55% moisture inhibited growth. Potassium and calcium lactate were as effective as the sodium salt in suppressing growth and aw.

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

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.

Viability of Listeria monocytogenes Strain Brie-1 in the Avian Egg.

The viability of Listeria monocytogenes strain Brie-1 was studied in raw and heat-treated (121°C, 15 min) whole eggs, albumen, or yolks during storage at 5 and 20°C. Studies with raw eggs showed that the organism grew only in egg yolks, where initial numbers (106 cells/g) increased to 108 cells/g (generation times of 1.7 d and 2.4 h at 5 and 20°C, respectively). Cell numbers in whole eggs initially declined and then leveled off. A sharp decline in cell numbers was observed in the raw albumen (to 102 cells/g after 22 d at 5°C and to <10 after 55 h at 20°C). In contrast, the organism grew in all heat-treated egg samples. Generation times for cooked whole eggs, yolks, and albumen were 1.9, 2.3, and 2.4 d at 5°C, and 2.6, 2.6, and 3.5 h at 20°C, respectively. The rapid initial decline in populations was observed in raw albumen (pH 8.9), and after adjustments to pH 7.0 or 8.0. Numbers of surviving cells/g after 35 d at 5°C were reduced to 104, 103, and <10, at pH 7, 8, and 9 respectively. With the exception of the raw albumen, refrigerated raw and cooked eggs supported survival and growth of L. monocytogenes , and hence can serve as vehicle of transmission of listeriosis.

Survival of Listeria monocytogenes in Ground Beef or Liver During Storage at 4 and 25°C.

Survival and growth of three Listeria monocytogenes strains (Scott A, Brie-1, and ATCC 35152) were studied in ground beef or liver during storage from freshness to spoilage at 4 and 25°C. Cells were enumerated on Plate Count Agar, Trypticase Soy Agar, and selective media, including McBride Listeria Agar (MLA), Cyclohexanedione Nalidixic Acid Phenylethanol Agar (CNPA), LiCl Phenylethanol Moxalactam Agar (LPM), and LPM with potassium tellurite (LPMT). Aerobic natural microflora in the fresh uninoculated samples ranged from 102 to 104 CFU/g, and L. monocytogenes inocula were ca. 103 or 105 CFU/g. Total aerobes after 2 weeks at 4°C were >108 or >107/g in meat or liver respectively, while recovered numbers of L. monocytogenes remained unchanged during a storage of over 30 d in either ground meat or liver. Samples stored at 25°C confirmed recovery but absence of multiplication of the organism. LPM or LPMT provided the best selective environment for direct plating of meat. Despite differences in composition and spoilage pattern of meat and liver, no difference was observed in the fate of L. monocytogenes in these foods.