CHARACTERIZATION OF A LACTIC ACID BACTERIUM,CARNOBACTERIUM PISCICOLA LK5, WITH ACTIVITY AGAINST LISTERIA MONOCYTOGENES AT REFRIGERATION TEMPERATURES1

A lactic acid bacterium (LK5) originally isolated from raw ground beef was characterized in relation to its ability to inhibit the growth of Listeria monocytogenes. The isolate, which was identified as Carnobacterium piscicola, inhibited the growth of 17 of 21 strains of Listeria (L. monocytogenes, L. innocua, L. ivanovii, L. welshimeri, and L. grayii). Its activity was not due to either acid or hydrogen peroxide production, but was related to the production of a heat stable bacteriocin. The isolate was most active against L. monocytogenes at refrigeration temperatures due to the combined effect of the pathogen's increased susceptibility, LK5's rapid growth rate, and enhanced bacteriocin production at low temperatures. Examination of the effect of inoculum ratios in co-cultures of C. piscicola LK5 and L. monocytogenes Scott A indicated that the lactic acid bacterium was active against L. monocytogenes even when the initial level of the pathogen was 100-fold greater. Evaluation of the impact of oxygen availability, initial pH, and sodium chloride content on the effectiveness of LK5 suggested that the isolate could be used to suppress the growth of Listeria in a variety of refrigerated foods.     

INHIBITION OF LISTERIA MONOCYTOGENES BY CARNOBACTERIUM PISCICOLA IN VACUUM-PACKAGED COOKED CHICKEN AT REFRIGERATION TEMPERATURES

Listeria monocytogenes inhibition by bacteriocinogenic Carnobacterium piscicola DX or nonbacteriocinogenic C. piscicola 2818 was examined in vacuum-packed minimally processed chicken cubes with gravy at 4, 8 and 15C. C. piscicola DX and C. piscicola 2818 at 10⁴ CFU/g were coinoculated individually with a pool of three Listeria monocytogenes strains at 10² CFU/g. At 4 and 8C , C. piscicola DX inhibited growth of L. monocytogenes by 3 logs, significantly (P < 0.05) more than did C. piscicola 2818. At 15C both C. piscicola strains inhibited L. monocytogenes by one log cycle. The pH of all inoculated systems decreased from an initial pH of 6.14 to final values ranging from 6.06 to 5.65 depending on the inocula used. Bacteriocin was detected in the systems coinoculated with C. piscicola DX. These studies demonstrate that lower temperatures and bacteriocin production enhanced L. monocytogenes inhibition by C. piscicola     

A MODEL FOR PREDICTING THE GROWTH OF LISTERIA MONOCYTOGENES IN PACKAGED WHOLE MILK

Experiments were conducted to determine growth characteristics of Listeria monocytogenes in sterilized whole milk at nine temperatures in the range of 277.15 to 308.15K (4 to 35C). Based on these data, the parameter values of the Baranyi dynamic growth model were statistically determined. Finite element software, ANSYS, was used to determine temperature distributions in milk cartons subject to a time‐varying ambient temperature profile. The space‐time‐temperature data were input to the Baranyi dynamic growth model, to predict the microbial population density distribution and the average population density in the milk carton. The Baranyi dynamic growth model and the finite element model were integrated and validated using experimental results from inoculated sterilized whole milk in half‐gallon laminated paper cartons. In all experiments, the milk cartons were subjected to the same temperature profile as the Baranyi dynamic growth model. Experimental microbial counts were within predicted upper and lower bounds obtained using the integrated Baranyi dynamic growth and finite element models. In addition, the growth curve at the mean value of initial physiological state parameter for L. monocytogenes underpredicted the microbial growth (standard error = 0.54 log (cfu/mL) and maximum relative difference = 15.49%).     

TEMPERATURE SHIFT EFFECTS ON INJURY AND DEATH IN LISTERIA MONOCYTOGENES SCOTT A

Exposure of Listeria monocytogenes Scott A grown at 37°C to a 1 h heat treatment at 52°C resulted in little death of the cells (< 0.5 log). However, as the temperature of growth decreased, there was an increase in the extent of death (> 4 logs at 10°C growth temperature). Heat induced injury, however, decreased as the growth temperature decreased. Shifting L. monocytogenes grown at 10, 19, or 28°C to 37°C for periods up to 5 h led to cells with increased heat tolerance. However, there was little effect on injury by the shift-up procedure. Presence of chloramphenicol during the shift-up period inhibited the gain in heat tolerance . L. monocytogenes grown at low temperatures (< 28°C), were more susceptible to killing by heat, but this susceptibility could be lost if cells grown at low temperatures are given a short incubation at 37°C. The data obtained here suggest that if foods containing L. monocytogenes are temperature-abused for even short periods, the organisms will acquire an increased heat tolerance and will require higher inactivation temperatures or longer processing times .     

MATHEMATICAL MODEL FOR THE SURVIVAL OF LISTERIA MONOCYTOGENES IN MEXICAN-STYLE SAUSAGE

Survival of Listeria monocytogenes in chorizos (Mexican‐style sausages) was modeled in relation to initial water activity (a_w0) and storage conditions using the Weibull cumulative distribution function. Twenty survival curves were generated from chorizos formulated at a_w0 = 0.85–0.97 then stored under four temperature (T) and air inflow velocity (F) conditions. The Weibull model parameters (α and β) were determined for every curve. Predicted survival curves agreed with experimental curves with R² = 0.945–0.992. Regression models (R² = 0.981–0.984) were developed to relate α and β to operating conditions. The times to one‐ and two‐log reduction in count (t_1D and t_2D) were derived from the Weibull model in terms of α and β. A parametric study revealed that L. monocytogenes survival was most sensitive to a_w0 between 0.90 and 0.95. The inactivation of L. monocytogenes could be maximized with higher T and lower a_w0; however, F did not significantly influence survival.     

METHODS FOR THE DETECTION OF FOODBORNE LISTERIA MONOCYTOGENES IN THE U.S.

A review of methods for the isolation and detection of Listeria monocytogenes used in the United States is presented. Methods reviewed include the cold enrichment technique, the FDA Method, the USDA Method, and two rapid techniques, the Listeria-Tek enzyme-linked immunosorbent assay and the Gene-Trak Listeria Assay. Comparisons of new rapid biochemical test kits, the MICRO-ID LISTERIA System, API Listeria, and the Rosco system vs. conventional tests of identification are also reviewed. Contemporary isolation methods detect all Listeria species so confirmation of L. monocytogenes is still necessary after isolation. The USDA method is the most practical of the cultural methods due to the rapid reporting of negative samples. Rapid methods (Listeria-Tek and the Gene-Trak Listeria Assay) are faster and more objective than cultural procedures but still depend on sample enrichment for detection of Listeria. These rapid techniques are best utilized when screening large numbers of food samples. All the rapid biochemical test kits reviewed provide fast reliable identification of Listeria species when compared to classical techniques. However, the API Listeria system identifies the test strains without a complementary CAMP test. Refinements are still needed in both cultural and rapid methods. Future Listeria methodology must emphasize molecular techniques not requiring enrichment which would be both rapid and specific for L. monocytogenes.     

GROWTH/SURVIVAL OF NATURAL FLORA AND LISTERIA MONOCYTOGENES ON REFRIGERATED UNCOOKED PORK AND TURKEY PACKAGED UNDER MODIFIED ATMOSPHERES

Listeria monocytogenes was inoculated onto fresh pork and turkey slices. Inoculated and control samples were packaged under modified atmospheres (100% N₂, and 20%/80% and 40%/60% CO₂O₂) or air in plastic bags of low gas permeability. Samples were stored at 1 and 7C. Samples stored in air showed a similar microbiological pattern to that usually observed in fresh meat stored aerobically. Packaging under modified atmospheres extended the meat shelf-life. Bacterial growth was strongly inhibited at 1C, particularly in samples stored under CO₂/O₂ enriched atmospheres. Temperature and pH were critical factors for L. monocytogenes growth. This pathogen grew only on pork (initial pH 5.3) packaged in air and stored at 7C. No L. monocytogenes growth was observed at 1C in any atmosphere assayed. However, growth on turkey (initial pH 6.0) was marked at 7C in all atmospheres tested, while at 1C, this bacterium grew weakly only on samples stored in air .     

A REVIEW OF METHODS FOR DETECTION OF THE PSYCHROTROPHIC FOODBORNE PATHOGENS LISTERIA MONOCYTOGENES AND AEROMONAS HYDROPHILA1

The detection of the psychrotrophic foodborne pathogens Listeria monocy-togenes and Aeromonas hydrophila in food depends on the use of various selective media designed specifically for their isolation. These selective media, which contain combinations of dyes, antibiotics, and other inhibitory substances, restrict the background microflora while permitting the desired organism (either L. monocytogenes or A. hydrophila) to form characteristic colonies. Since the selective media are not completely specific, confirmation tests specific to L. monocytogenes or A. hydrophila are used to verify the identity of the respective isolates. It has been observed that the inhibitory substances used will not permit injured (stressed) cells to form colonies and special techniques are needed to recover injured cells. The present techniques, while not ideal, do allow for a reasonably quantitative estimate of any L. monocytogenes or A. hydrophila present in a food.     

A COMPARISON BETWEEN TWO METHODS FOR THE RECOVERY OF LISTERIA MONOCYTOGENES IN MILK POWDER REFERENCE SAMPLES

Two methods (A and B) for the recovery of Listeria monocytogenes were compared using reference samples based on spray drying of milk containing this pathogen and competitive microflora. For method A, the sample was inoculated in a buffered liquid medium (same as IDF Pre-enrichment Broth) and then passed to IDF Enrichment Broth plus phosphate buffer. For method B, the sample was inoculated in FDA Enrichment Broth (LEB) and then passed to LEB (subenrichment). The solid selective medium used in both cases was Listeria Selective Agar (Oxford formulation). The sensitivity and specificity of method B was 95.2% and 100% vs. 76.1% and 88.8% for method A, respectively.     

INCIDENCE, SURVIVAL AND GROWTH OF LISTERIA MONOCYTOGENES IN READY-TO-USE MIXED VEGETABLE SALADS IN SPAIN

A total of 116 commercial samples of mixed vegetable salads, packaged in plastic bags, were examined for the presence of Listeria monocytogenes during storage at 4C. Commercial products, belonging to 4 different batches, were sampled over a period of one year and stored for 300 h at 4C in the laboratory. Different sets of enrichment and plating media were used to recover organisms, with subsequent identification by Pasco System and serotyping techniques. Out of a total of 70 control (noninoculated) samples, 21 (30%) were observed to contain Listeria monocytogenes, belonging to serotypes 3a and 3b. A study of salad inoculated with 10³ cfu/g Listeria monocytogenes showed that the initial inoculum increased less than tenfold over the experimental period (300 h). During storage of the product, CO₂ reached nearly 30% and O₂ was no longer detected at 60 h, due to the respiration of the vegetables. The pH inside the packages remained around 6. The specific growth rate in the salad was calculated using the Dmodel Program, which gave a rate of 0.003h^-1, a lower figure than that reported by other authors. This study found growth patterns different to those previously reported for salads with separate ingredients. Our results agree with previous reports that modified atmosphere does not greatly inhibit Listeria monocytogenes. This reflects a lower specific growth rate in this food compared to media, and illustrates the difficulty of validating, in complex food systems, mathematic models based on culture media .     

EFFECT OF STORAGE TEMPERATURE ON THE GROWTH OF LISTERIA MONOCYTOGENES ON QUESO BLANCO SLICES*

A five‐strain cocktail of Listeria monocytogenes (10⁴ cfu/mL) was inoculated onto individual vacuum‐packaged slices (ca. 50 g each) of a commercial, Hispanic‐style cheese, that being Queso Blanco. Growth was determined at appropriate intervals during storage at 5, 10, 15, 20 and 25C. In general, as the incubation temperature increased, a shorter lag phase duration (LPD) and a faster growth rate (GR) were observed. The LPD values at 5, 10, 15, 20 and 25C were 65.3, 19.9, 2.1, 8.4 and 11.4 h, respectively. The GR values were 0.011, 0.036, 0.061, 0.090 and 0.099 log cfu/h at 5, 10, 15, 20 and 25C, respectively. There were no statistical differences in LPD at 10, 15, 20 and 25C. However, the LPD during growth at 5C was statistically (P ≤ 0.05) longer than at all other temperatures. The GR values at 20 and 25C were not significantly different from each other, whereas the GR values at 5, 10 and 15C were significantly different from each other as well as from the GR at 20 and 25C (P ≤ 0.05). The maximum population density (MPD) showed relatively little variation over the range of storage temperatures tested, with an average of 8.38 log cfu/g (SD = 0.33). The results of this study indicate that not even the lowest trial temperature of 5C prevented growth over time of the inoculated L. monocytogenes on this sliced product, and that proper storage and handling procedures are required to prevent the bacterium from contaminating the product and/or to control its growth.     

EXTINCTION OF LISTERIA MONOCYTOGENES IN SINGLE-STRENGTH ORANGE JUICE: COMPARISON OF METHODS FOR DETECTION IN MIXED POPULATIONS1

The FDA method of selective enrichment followed by selective plating on modified McBride agar was capable of detecting the presence of L. monocytogenes inoculated into a typical, commerical, reconstituted, single-strength orange juice at the 10⁰ cfu/mL level. The KOH shock treatment, formerly recommended in the FDA protocol, negatively affected recovery of Listeria at 10⁰ and 10¹ levels in juice which also had a low background microflora (10² cfu/mL total count); however, KOH treatment was required for Listeria detection in juice which had high background microflora (10⁸ cfu/mL). Two other protocols for detection of Listeria which utilized cold enrichment or different selective enrichment media were less effective than the FDA procedure due to heavy growth of background microflora. No Listeria was detected in 100 commerical orange juice samples from dairy and nondairy processors in geographically distinct areas of North America using the FDA method.     

THE EFFECT OF AEROBIC MESOPHILIC MICROFLORAL LEVELS ON THE ISOLATION OF INOCULATED LISTERIA MONOCYTOGENES STRAIN LM82 FROM SELECTED FOODS

The effect of aerobic mesophilic microfloral concentration on the isolation of Listeria monocytogenes LM82 was studied in 31 (18 cheeses and 7 noncheese) retail foods having standard plate counts of 10¹ to 10⁸ colony forming units (CFU)/g. Foods were spiked with L. monocytogenes and enriched at 30°C for 24 h in a selective enrichment broth used in a U.S. Food and Drug Administration method. Inoculum levels for isolation on modified McBride agar ranged from 0.1 to > 5 × 10³ with a geometric mean value of 5 inoculated CFU/g or 1.4 CFU/g. Pure Enterococcus (Streptococcus) faecalis ( 0 to 6 × 10⁶ inoculated CFU/mL ) in the absence of food matrix had no effect on the enrichment of L. monocytogenes. Ease of isolation of LM82 was independent of the food microflora concentration both generally and in the specific food type of 9 Brie cheeses. Competition, when it occurs, therefore, may be due to specific bacterial competitors rather than bacterial numbers .     

EFFECT OF pH, ACIDULANT, SODIUM CHLORIDE AND TEMPERATURE ON THE GROWTH OF LISTERIA MONOCYTOGENES

Growth of two Listeria monocytogenes strains in tryptic soy broth containing NaCI or combinations of NaCI and acidulants at different pHs and temperatures was investigated . L. monocytogenes was capable of growing in 10% NaCI at 35°C and 12% NaCI at 25°C and 10°C. The maximum NaCI for growth changed when NaCI and pH, in combination with different acidulants and temperature, were tested. The minimum pH/salt level for initiation of growth of L. monocytogenes ranged from 5.0–5.6/8–10% at 35°C and 25°C and 5.6/8% at 10°C, depending upon the acidulant and the strain. Greatest antimicrobial activity occurred at 35°C. Greatest survival occurred at 10°C. In this study L. monocytogenes appeared to persist and tolerate a combination of low pH, high salt and low temperatures .     

EFFICACY OF PULSED ELECTRIC FIELDS FOR LISTERIA MONOCYTOGENES INACTIVATION AND CONTROL IN HORCHATA

Although Listeria monocytogenes is readily destroyed by thermal treatment, the factor that makes it particularly difficult to control in nonpasteurized foods is its ability to grow at refrigeration temperatures. In heat‐sensitive products, nonthermal technologies such as pulsed electric fields (PEF) as part of hurdle technology could minimize the presence of foodborne pathogens. The influence of PEF‐treatment conditions, inoculum size and substrate conditions on the inactivation and recovery of L. monocytogenes in a traditional low‐acid, vegetable beverage was investigated. The combined effect of PEF, low temperature (5C) and low inoculum level contributed to slow down the recovery of sublethally injured cells. However, at 12 or 16C, this elongation of the lag phases after PEF treatment observed for low inoculum levels of cells was not achieved. Therefore, to prevent the development of L. monocytogenes in low‐acid products by PEF, it may be necessary to combine it with low refrigeration temperatures during distribution and storage, as well as to achieve a very low initial contamination by pathogens in the raw ingredients.     

食品中单增李斯特菌的存在现状及检测方法研究进展

单核细胞增生性李斯特菌（简称单增李斯特菌）是常见的食源性致病菌,该菌可广泛存在于多种食品中,如肉制品、奶制品、水产品、蔬菜等,该菌在欧美国家曾导致多次的食物中毒的爆发,因此对食品中单增李斯特菌的快速检测非常必要,就单增李斯特菌在食品中的污染情况和检测方法作一综述。