Growth rate and growth probability of Listeria monocytogenes in dairy, meat and seafood products in suboptimal conditions

AIMS: To evaluate the performances of models predicting the growth rate or the growth probability of Listeria monocytogenes in food. METHODS AND RESULTS: Cardinal and square root type models including or not interactions between environmental factors and probability models were evaluated for their ability to describe the behaviour of L. monocytogenes in liquid dairy products, cheese, meat and seafood products. Models excluding interactions seemed sufficient to predict the growth rate of L. monocytogenes. However, the accurate prediction of growth/no-growth limits needed to take interactions into account. A complete and a simplified form (preservatives deducted) of a new cardinal model including interactions and parameter values were suggested to predict confidence limits for the growth rate of L. monocytogenes in food. This model could also be used for the growth probability prediction. CONCLUSIONS: The new cardinal model including interactions was efficient to predict confidence limits for the growth rate of L. monocytogenes and its growth probability in liquid dairy products, meat and seafood products. In cheese, the model was efficient to predict the absence of growth of the pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: The suggested model can be used for risk assessment and risk management concerning L. monocytogenes in dairy, meat and seafood products.     

Antibacterial activity of pepsin-digested lactoferrin on foodborne pathogens in buffered broth systems and ultra-high temperature milk with EDTA

AIMS: To evaluate the antimicrobial activity in peptone yeast extract glucose (PYG) broth and ultra-high temperature (UHT) milk of bovine lactoferrin hydrolysate (LFH) with pepsin against the foodborne pathogens Salmonella Stanley, Escherichia coli, Listeria monocytogenes and Staphylococcus aureus. METHODS AND RESULTS: The LFH was suspended in PYG and the minimum inhibitory concentration for each pathogen determined. The LFH was also suspended in UHT milk adjusted to pH 4 or 7, samples incubated at 4 or 35 degrees C and the change in bacterial cell population determined. Experiments in UHT milk were conducted using L. monocytogenes and E. coli O157:H7. At pH 4 LFH reduced the population of E. coli O157:H7 and L. monocytogenes by approx. 2 log; however, only E. coli O157:H7 was inhibited in samples adjusted to pH 7. The addition of EDTA (10 mg ml(-1)) to UHT milk supplemented with LFH did not markedly influence the growth of E. coli O157:H7 or L. monocytogenes. CONCLUSIONS: The results suggest that, under low pH and refrigeration conditions, LFH can limit the growth or reduce the population of pathogenic bacteria in a dairy product. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural preservatives that are active against Gram-negative and Gram-positive bacteria are desirable to the food industry. This study demonstrates that LFH is effective in a complex food system. Moreover, the LFH used was not purified, making its use by industry more attractive.     

Fate of Listeria monocytogenes during manufacture and ripening of semi-hard cheese

Semi-hard cheeses were experimentally elaborated with pasteurized milk from sheep, goat and cow (15: 35: 50) and inoculated to contain 1.9 times 10⁵ Listeria monocytogenes /ml in cheeses 1 and 2 and 4 times 10³ L. monocytogenes /ml in cheeses 3 and 4. Counts of L. monocytogenes were determined by direct surface plating of samples on listeria selective agar medium. The results show the substantial survival of L. monocytogenes present in milk during manufacture and ripening of this type of cheese.     

Survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer

The survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer was investigated. Raw milk was inoculated with different initial concentrations of L. monocytogenes and heated at temperatures ranging from 69 degrees to 73 degrees C. Listerias were not isolated from any of the milk samples immediately after thermal treatment. They were isolated, however, from 46.6% of heated samples (none from samples heated at 73 degrees C) after variable periods at refrigeration temperature. The results suggest that a low number of listerias survive some thermal treatments, but a cold enrichment is necessary to repair the thermally injured cells and detect these organisms in milk. The importance of the isolation technique in the recovery of listerias from pasteurized milk samples is discussed.     

Survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer

The survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer was investigated. Raw milk was inoculated with different initial concentrations of L. monocytogenes and heated at temperatures ranging from 69° to 73°C. Listerias were not isolated from any of the milk samples immediately after thermal treatment. They were isolated, however, from 46.6% of heated samples (none from samples heated at 73°C) after variable periods at refrigeration temperature. The results suggest that a low number of listerias survive some thermal treatments, but a cold enrichment is necessary to repair the thermally injured cells and detect these organisms in milk. The importance of the isolation technique in the recovery of listerias from pasteurized milk samples is discussed.     

Occurrence of Listeria species in raw milk and dairy products produced in Northern Ireland.

The overall incidence of Listeria spp. in raw milk samples surveyed was found to be 25.0% (Listeria monocytogenes 15.3%), with the incidence in samples from processing centres 54.0% (L. monocytogenes 33.3%); this was higher than that in samples from dairy farms (Listeria spp. 8.8%; L. monocytogenes 5.3%). The FDA enrichment procedure was much more productive than cold enrichment and Oxford agar was superior to modified McBride agar for isolation of Listeria. Listeria monocytogenes was never isolated by direct plating of raw milk samples on Oxford agar at a detection level of 1.0 cfu/ml. Listeria spp. were isolated from 1 of 95 pasteurized milk samples (L. monocytogenes) and 1 of 33 soft cheese samples (L. seeligeri). Restriction fragment length polymorphism was more useful than sero- or phage-typing for typing of L. monocytogenes strains, and results suggest that specific L. monocytogenes strains may persist in both farm and processing environments.     

Occurrence of Listeria species in raw milk and dairy products produced in Northern Ireland

J. HARVEY AND A. GILMOUR. 1992. The overall incidence of Listeria spp. in raw milk samples surveyed was found to be 25.0% ( Listeria monocytogenes 15.3%), with the incidence in samples from processing centres 54.0% ( L. monocytogenes 33.3%); this was higher than that in samples from dairy farms ( Listeria spp. 8.8% L. monocytogenes 5.3%). The FDA enrichment procedure was much more productive than cold enrichment and Oxford agar was superior to modified McBride agar for isolation of Listeria. Listeria monocytogenes was never isolated by direct plating of raw milk samples on Oxford agar at a detection level of 1.0 cfu/ml. Listeria spp. were isolated from 1 of 95 pasteurized milk samples ( L. monocytogenes ) and 1 of 33 soft cheese samples ( L. seeligeri ). Restriction fragment length polymorphism was more useful than sero- or phage-typing for typing of L. monocytogenes strains, and results suggest that specific L. monocytogenes strains may persist in both farm and processing environments.     

Listeria monocytogenes in pasteurized milk

An haemolytic Listeria monocytogenes strain pathogenic to mice was isolated from 6 out of 28 (21.4%) pasteurized milk samples (3.2% fat milk treated at 78 degrees C for 15 s) marketed by a Madrid processing plant. Listeria grayi was recovered from 25 of the samples (89.2%) and L. innocua from 3 samples (10.7%). One milk sample was contaminated with L. welshimeri. No strains of L. ivanovii, L. seeligeri, L. murrayi, or L. denitrificans were isolated. These results show that pathogenic Listeria strains can be isolated from pasteurized milk and reinforce the hypothesis that this food product may be the source of numerous human listeriosis.     

Survival of Escherichia coli O157:H7, Listeria monocytogenes 4b and Yersinia enterocolitica O3 in different yogurt and kefir combinations as prefermentation contaminant

AIMS: To compare microbiological safety of yogurt, kefir and different combinations of yogurt and kefir samples by using three foodborne pathogenic strains (Escherichia coli O157:H7, Listeria monocytogenes 4b and Yersinia enterocolitica O3) as indicators. METHODS AND RESULTS: Fresh yogurt and kefir drinks were added to pasteurized milk at a 5% rate either separately or together, and then incubated at different temperatures (43 degrees C for yogurt and 30 degrees C for kefir), depending on appropriate growth temperature of their starter microflora. While traditional yogurt was found to be the least suppressive on the three pathogenic micro-organisms, samples obtained from two subsequent fermentation process (samples fermented at 43 degrees C for 3 h and at 30 degrees C for 21 h) were more suppressive than that of traditional kefir. There was no significant survival difference between E. coli O157:H7 and L. monocytogenes 4b in samples tested (P > 0.05), but Y. enterocolitica O3 was more susceptible than other two test strains (P < 0.05). CONCLUSIONS: The microbiological safety of the dairy product fermented at two consecutive periods was superior than that of traditional yogurt or kefir alone. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments may mimic what happens when yogurt and kefir starter micro-organisms are combined in a milk fermentation process with different time and temperature periods.     

Probabilistic Model for Listeria monocytogenes Growth during Distribution,Retail Storage,and Domestic Storage of Pasteurized Milk

A survey on the time-temperature conditions of pasteurized milk in Greece during transportation to retail, retail storage, and domestic storage and handling was performed. The data derived from the survey were described with appropriate probability distributions and introduced into a growth model of Listeria monocytogenes in pasteurized milk which was appropriately modified for taking into account strain variability. Based on the above components, a probabilistic model was applied to evaluate the growth of L. monocytogenes during the chill chain of pasteurized milk using a Monte Carlo simulation. The model predicted that, in 44.8% of the milk cartons released in the market, the pathogen will grow until the time of consumption. For these products the estimated mean total growth of L. monocytogenes during transportation, retail storage, and domestic storage was 0.93 log CFU, with 95th and 99th percentiles of 2.68 and 4.01 log CFU, respectively. Although based on EU regulation 2073/2005 pasteurized milk produced in Greece belongs to the category of products that do not allow the growth of L. monocytogenes due to a shelf life (defined by law) of 5 days, the above results show that this shelf life limit cannot prevent L. monocytogenes from growing under the current chill chain conditions. The predicted percentage of milk cartons—initially contaminated with 1 cell/1-liter carton—in which the pathogen exceeds the safety criterion of 100 cells/ml at the time of consumption was 0.14%. The probabilistic model was used for an importance analysis of the chill chain factors, using rank order correlation, while selected intervention and shelf life increase scenarios were evaluated. The results showed that simple interventions, such as excluding the door shelf from the domestic storage of pasteurized milk, can effectively reduce the growth of the pathogen. The door shelf was found to be the warmest position in domestic refrigerators, and it was most frequently used by the consumers for domestic storage of pasteurized milk. Furthermore, the model predicted that a combination of this intervention with a decrease of the mean temperature of domestic refrigerators by 2°C may allow an extension of pasteurized milk shelf life from 5 to 7 days without affecting the current consumer exposure to L. monocytogenes.L. monocytogenes is an important safety concern for the dairy industry. Several listeriosis outbreaks have been associated with the consumption of dairy products, including pasteurized milk (13, 22). An effective control of L. monocytogenes in pasteurized milk should be based on the selection of raw milk and the controls of the processing, packaging, distribution, and storage conditions. In general, the pathogen is effectively controlled during pasteurization. However, its presence in the finished product is possible as a result of postpasteurization contamination from sources in the plant environment. Considering that the levels of postpasteurization contamination are usually very low, the extent of L. monocytogenes growth during distribution, retail storage, and domestic storage is of major significance for the safety status of pasteurized milk at the time of consumption.The growth of L. monocytogenes during distribution and storage of pasteurized milk can be evaluated using the available predictive models. During the last decade, a large number of mathematical models for L. monocytogenes growth have been published (9, 11, 16, 19, 21, 24, 26, 31, 38), and some of them have been targeted to pasteurized milk (1, 40, 49). However, since the available data show that conditions that prevail during the chill chain vary significantly (8, 17, 23, 27, 28, 34, 44, 45, 48), the value of a deterministic application of these models as a tool in safety management of pasteurized milk would be limited.In recent years the need for taking into account the variabilities of the various factors in predictive microbiology has been increasingly recognized, leading to a more sophisticated modeling approach called probabilistic or stochastic modeling. The main characteristic of probabilistic modeling is the specific quantification of variabilities using probability distributions for the input data rather than point estimates. The importance of characterizing variability was stressed by Nauta (41), who illustrated the differences in decisions that could result if variability is ignored. Probabilistic modeling is being used with increasing frequency in the area of food safety. It has been extensively applied in quantitative microbial risk assessments (12, 18, 20), in quality and safety management systems (25, 30, 32), and recently for more specific topics, such as the evaluation of the effects of food processing (39) and the compliance of food products to safety criteria set by regulations (33).In the present study a probabilistic modeling approach was applied for evaluating the growth of Listeria monocytogenes in pasteurized milk from production to the time of consumption based on a Monte Carlo simulation. The objectives of the study were (i) to estimate the growth of the pathogen at the various stages of the chill chain, including transportation to retail, retail storage, and domestic storage, (ii) to analyze the importance of the chill chain factors, (iii) to evaluate the effects of selected intervention scenarios related to the improvement of the chill chain and handling conditions, and (iv) to evaluate the effect of a potential extension of milk shelf life on the growth of Listeria monocytogenes.     

Single cell variability of L. monocytogenes grown on liver pâté and cooked ham at 7 degrees C: comparing challenge test data to predictive simulations

AIMS: The variability in growth between individual Listeria monocytogenes cells was investigated on liver paté and cooked ham. These results were compared to Monte Carlo simulations based on data collected previously in broths (Francois et al., submitted for publication). METHODS AND RESULTS: Single cells were isolated by a dilution protocol and inoculated on 15 g samples of liver paté and cooked ham, pasteurized in the packaging. Of each product, 250 samples were inoculated, of which 50 samples were analysed for L. monocytogenes on each analysis day. Results were compared to simulations, based on distributions that describe the variability of the individual cell lag phases and generation times of L. monocytogenes cultivated in broths. Based on the same simulation techniques, the variability effect was investigated for different inoculum levels (10, 100, 10,00 and 10,000 cells). It was demonstrated that the expected variability of the outgrowth of L. monocytogenes in a challenge test is very high for low inoculum levels. CONCLUSIONS: The variability in growth characteristics observed between different single L. monocytogenes cells on foods is very large. The simulations based on the previously collected optical density data in broths, could be confirmed by foods inoculated with single L. monocytogenes cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The large variability between different individual L. monocytogenes cells has serious consequences for the experimental design of a challenge test. One thousand cells per portion are necessary in order to reduce the variability to acceptable levels and quantify the behaviour of the pathogen consistently with a reasonable number of challenge tests.     

Comparison of a quadratic response surface model and a square root model for predicting the growth rate of Yersinia enterocolitic

Polynomial equations, relating the growth rate of Yersinia enterocolitica to temperature (0–25°C) and pH (4.5–6-5) in a liquid medium were constructed for four different acidulants. The logarithm of the time for a 100-fold increase in bacterial numbers could be represented by a quadratic response surface function of pH and temperature. The interactions between pH and temperature on growth rate were found to be additive. Values for a 2 log cycle increase in growth derived from the model were in good agreement with experimental values. Predictions from the quadratic model and from a square root model were compared with experimental values in laboratory media and UHT milk. The mean square error (MSE) for the quadratic response surface model was smaller than that for the square root model in 81% of cases. In UHT milk the square root model increasingly underestimated growth rate, as the temperature decreased and would 'fail dangerous' if used for predictive purposes. This indicated that the response surface model is more reliable for predicting the growth of Y. enterocolitica under conditions of sub-optimal temperature and pH.     

Osmoprotectants and cryoprotectants for Listeria monocytogenes

Listeria monocytogenes is a foodborne pathogen that can grow in high osmotic strength environments and at refrigeration temperatures. Glycine betaine, proline betaine, acetylcarnitine, carnitine, gamma-butyrobetaine and 3-dimethylsulphoniopropionate all acted as osmoprotectants, as evidenced by an increase in growth rate of L. monocytogenes 10403S and Scott A when provided with these compounds, while being stressed in defined medium containing 0.7 M NaCl. These same compounds exhibited cryoprotective activity, as evidenced by increasing the growth rate of L. monocytogenes at 5 degrees C. Ectoine, hydroxy ectoine, pipecolic acid and proline were ineffective as osmoprotectants or cryoprotectants under these conditions. The presence of osmoprotectants and cryoprotectants in foods may provide compounds assisting L. monocytogenes to overcome the barriers of high osmotic strength and low temperature that otherwise control microbial growth.     

Physiological evaluation of stimulated growth of Listeria monocytogenes by Pseudomonas species in milk

Experiments were designed to elucidate a possible physiological mechanism for stimulated growth of Listeria monocytogenes by Pseudomonas fluorescens in milk. The ability of L. monocytogenes to grow in milk under aerobic and microaerophilic environments was compared. Neither environment favored the growth of the organism at 10 degrees C. Autoclaved whole milk was inoculated with P. fluorescens P26 and preincubated for 3 days at 10 degrees C followed by inoculation with L. monocytogenes Scott A and further incubation for 8 days at 10 degrees C. Changes in selected milk components were monitored over the 8-day period. The amount of lactose in the milk was determined, as well as the extent of proteolysis and lipolysis. Both L. monocytogenes and P. fluorescens were able to hydrolyze milk fat but were unable to use lactose. Milk protein was hydrolyzed by P. fluorescens but not by L. monocytogenes. Whole milk partially proteolyzed by treatment with purified protease was inoculated with L. monocytogenes. Results indicated that the growth of L. monocytogenes was stimulated in proteolyzed milk. This is the first report to provide evidence describing a likely mechanism for commensalism between L. monocytogenes and Pseudomonas spp.     

Growth, survival, proliferation and pathogenesis of Listeria monocytogenes under low oxygen or anaerobic conditions: a review

Listeria monocytogenes is a Gram positive facultative anaerobe that causes listeriosis, a disease that mainly affects the immune-compromised, the elderly, infants and pregnant women. In the susceptible immune challenged population, listeriosis is very severe and has a fatality rate of up to 30%. Control of L. monocytogenes is difficult due to its: 1) widespread presence in the environment, 2) intrinsic physiological resistance, 3) ability to adapt to external stresses and 4) ability to grow at a wide range of temperatures. L. monocytogenes encounters anaerobic conditions in the external environment as well as during pathogenesis. Although L. monocytogenes is a facultative anaerobe, the differential effects of O(2) and oxidation-reduction potential on the multiplication of L. monocytogenes have not been established. In addition, most laboratory studies to determine the growth, survival and persistence of this pathogen in foods as well as in the environment have emphasized the response of this pathogen under aerobic conditions. Consequently, this has led to a limited understanding of the metabolic and physiological responses of L. monocytogenes in low oxygen environments. Therefore, the objective of our review was to highlight the progress that has been made in L. monocytogenes research with emphasis on the role of low oxygen and/or anaerobiosis in the growth, survival and proliferation of this pathogen in the environment as well as during pathogenesis.     

Natural milk fatty acids affect survival and invasiveness of Listeria monocytogenes

The effects of 12 fatty acids, naturally occurring in milk from several mammalian species, on the survival and invasion ability of Listeria monocytogenes , a food-borne pathogen, were determined. The survival was tested in the presence of 200 μg ml⁻¹ fatty acids suspended in brain hearth infusion broth or in storage conditioning solution (NaCl 1%) of Mozzarella cheese, an Italian soft unripened cheese, at pH 7·0 or 5·0. Lauric (C12:0), linoleic (C18:2) and linolenic (C18:3) acids exerted the strongest bactericidal activity. The invasive efficiency of L. monocytogenes , determined in the Caco-2 enterocyte-like cell line, was strongly decreased in the presence of the fatty acids tested (from about 20 to 500-fold). This research suggests that naturally occurring fatty acids of milk, supplemented in milk derivatives, could affect both bacterial growth and invasiveness and consequently, could serve as barriers towards L. monocytogenes infection.     

THE RATES OF GROWTH OF SOME THERMODURIC BACTERIA IN PURE CULTURE AND THEIR EFFECTS ON TESTS FOR THE KEEPING QUALITY OF MILK

SUMMARY: The growth rates of eleven representative thermoduric bacteria, comprising 3 aerobic spore formers, 3 streptococci, 1 Corynebacterium lacticum and 4 micrococci, have been determined in glucose broth and sterile pasteurized milk at 37·5°, 26° and 15°. The spore formers and streptococci were generally not affected by the presence of inhibitory factors in pasteurized milk. When multiplication of micrococci and C. lacticum occurred in milk this was only after a lag period. One micrococcus showed an unusual series of growth phases in glucose broth at 37·5°, possibly due to the appearance of mutants or to adaptation of the organism to growth at that temperature. This was not observed in pasteurized milk. C. lacticum died off when incubated in glucose broth at 37·5°.
None of the keeping quality tests was more effective than any other in detecting these organisms in milk. The micrococci and C. lacticum had little effect on the keeping quality of pasteurized milk within the period of 'commercial life'. Some of the spore formers and streptococci showed marked differences in the end-points with the clot-on-boiling and the alcohol precipitation tests.     

Sensitization of heat-treated Listeria monocytogenes to added lysozyme in milk.

Listeria monocytogenes was highly resistant to hen egg white lysozyme in whole milk but was sensitive in media and in phosphate buffer. Methods to sensitize the pathogen to lysozyme in milk were investigated. Treatment of whole milk by cation exchange to remove minerals, particularly Ca2+ and Mg2+, slightly promoted inactivation of L. monocytogenes by lysozyme at 4 degrees C over a period of 6 days. Heat treatment (62.5 degrees C for 15 s) strongly sensitized L. monocytogenes to lysozyme in demineralized milk and in MES [2-(N-morpholino)ethanesulfonic acid] buffer. Addition of Ca2+ or Mg2+ to the demineralized milk restored resistance to lysozyme. Cells were more rapidly heat inactivated at 55 degrees C in demineralized milk containing lysozyme, and addition of Ca2+ to the demineralized milk restored the resistance to heat. The results indicate that minerals or mineral-associated components protect L. monocytogenes from inactivation by lysozyme and heat in milk, probably by increasing cell surface stability. The heat treatment of foods containing added lysozyme can probably play a significant role in producing microbiologically safe foods.