Listeria Monocytogenes In Milk Research Articles

Comparison of growth limits of Listeria monocytogenes in milk, broth and cheese

To determine growth initiation differences of Listeria monocytogenes between a cheesemaking context, milk and tryptic soy broth (TSB). A laboratory-scale cheese was made with a mix of two strains of L. monocytogenes at four initial pH values, five water activity (a(w)) values and two contamination levels at 30°C. Counts of L. monocytogenes were determined at time 0 and after 8h of cheese manufacture. Milk and TSB at the same pH and a(w) conditions were inoculated with the L. monocytogenes mix in multi-well plates. Growth was determined by plating each well onto Agosti & Ottaviani Listeria Agar after 8h of incubation at 30°C. Each condition was repeated six times, and growth initiation probability was modelled with logistic regression models. Growth initiation boundaries were obtained for each matrix type. The results showed that the growth limits were matrix dependent. In the three matrix types, a(w) was the most important factor affecting the probability of growth initiation. Contamination level affected growth TSB and cheesemaking conditions. The interface wideness and position in cheese, milk and TSB were dissimilar, indicating that the use of models evaluated in TSB or milk could not be used to predict the behaviour of L. monocytogenes under cheesemaking conditions. Predictive models generated in liquid media are not necessarily adaptable to solid food, and the generation of real food models is necessary.

Effect of growth and recovery temperatures on pressure resistance of Listeria monocytogenes

Experimental conditions can affect the outcome of bacterial stress-tolerance assays. Growth conditions that optimize microbial recovery should be established to help evaluate the effectiveness of treatment conditions for food safety. The objectives of this study were to determine the effects of growth and recovery temperatures on pressure resistance of early stationary-phase Listeria monocytogenes in milk. The tested conditions were the following: (1) L. monocytogenes was grown at various temperatures (10, 15, 20, 25, 30, 35, 40 and 43 °C), suspended in ultra-high temperature (UHT) -processed whole milk, pressure-treated at 400 MPa for 2 min at 21 °C and recovered on Tryptic Soy Agar supplemented with 0.6% yeast extract (TSAYE) at 35 °C; (2) L. monocytogenes was grown at 35 and 43 °C, pressure treated in milk (400 and 500 MPa, respectively, for 2 min at 21 °C) and recovered on TSAYE at various temperatures (4, 10, 15, 20, 25, 30, 35 and 40 °C); (3) L. monocytogenes originally grown at 35 °C, was pressure treated in milk (400 or 450 MPa for 2 min at 21 °C), and recovered on TSAYE at 10 °C for various time intervals (1, 2, 3, 6, 9 and 12 days) then at 35 °C for 5 days. There was no significant difference ( P > 0.05) in pressure-resistance of L. monocytogenes grown at 10 to 25 °C with approximately 6.5-log CFU/ml population reductions. At growth temperatures greater than 25 °C, pressure resistance increased with less than 1-log CFU/ml reduction observed for L. monocytogenes originally grown at 43 °C. After pressure treatment, regardless of growth temperature and pressure treatment, the greatest recovery of L. monocytogenes was within the 4 to 20 °C range; maximum recovery at 10 °C required approximately 24 days. The time for comparable post-pressure treatment recovery could be reduced by incubation at 10 °C for at least 2 days followed by incubation at 35 °C for 5 days. The findings of the present study indicate that growth and recovery temperatures affect the pressure resistance of L. monocytogenes and should, therefore, be taken into account when assessing the adequacy of inactivation treatments.

Fat content increases the lethality of ultra-high-pressure homogenization on Listeria monocytogenes in milk

Listeria monocytogenes CCUG 15526 was inoculated at a concentration of approximately 7.0log10cfu/mL in milk samples with 0.3, 3.6, 10, and 15% fat contents. Milk samples with 0.3 and 3.6% fat content were also inoculated with a lower load of approximately 3.0log10cfu/mL. Inoculated milk samples were subjected to a single cycle of ultra-high-pressure homogenization (UHPH) treatment at 200, 300, and 400MPa. Microbiological analyses were performed 2h after the UHPH treatments and after 5, 8, and 15 d of storage at 4°C. Maximum lethality values were observed in samples treated at 400MPa with 15 and 10% fat (7.95 and 7.46log10cfu/mL), respectively. However, in skimmed and 3.6% fat milk samples, complete inactivation was not achieved and, during the subsequent 15 d of storage at 4°C, L. monocytogenes was able to recover and replicate until achieving initial counts. In milk samples with 10 and 15% fat, L. monocytogenes recovered to the level of initial counts only in the milk samples treated at 200MPa but not in the milk samples treated at 300 and 400MPa. When the load of L. monocytogenes was approximately 3.0log10cfu/mL in milk samples with 0.3 and 3.6% fat, complete inactivation was not achieved and L. monocytogenes was able to recover and grow during the subsequent cold storage. Fat content increased the maximum temperature reached during UHPH treatment; this could have contributed to the lethal effect achieved, but the amount of fat of the milk had a stronger effect than the temperature on obtaining a higher death rate of L. monocytogenes.

Effect of nanovesicle-encapsulated nisin on growth of Listeria monocytogenes in milk

Commercial nisin was encapsulated in nanovesicles (mean diameter 140 nm) prepared from partially purified soy lecithin. Nisin-loaded liposomes and unencapsulated (free) nisin were initially tested in BHI medium and skim milk inoculated with Listeria monocytogenes and incubated for 48 h at 30 °C. At such abuse temperature conditions, free nisin showed better inhibitory than the liposomal counterparts. Subsequently, the effect of encapsulated or free nisin was evaluated in combination with refrigeration (7 ± 1 °C) in both whole (3.25% fat) and skim (0% fat) milk for up to 14 day. A decrease of 3–4 log cycles in L. monocytogenes counts was observed for free and encapsulated nisin at 0.5 mg/ml concentration. Liposome encapsulation of antimicrobial peptides may be important to overcome stability issues and interaction with food components. The utilization of nanovesicle-encapsulated nisin in combination with low temperatures appeared to be effective to control L. monocytogenes in milk, emphasizing the importance of hurdle technology to assure food safety.

Rapid On-chip flow Cytometric Detection of Listeria monocytogenes in Milk

Listeria monocytogenes (L. monocytogenes) is a Gram-positive, intracellular bacterium that can cause severe infections in humans. Contamination by L. monocytogenes in foods represents a potential public health problem. Here we describe a rapid method for the detection of L. monocytogenes in milk by flow cytometry based on microfluidics (on-chip flow cytometry). This rapid identification of L. monocytogenes is based on fluorescence in situ hybridization (FISH) with a Cy5-labeled rRNA-targeting oligonucleotide probe. FISH experiments were successfully analyzed using a commercially available on-chip flow cytometry. FISH results with bacterial cultures indicated that the L. monocytogenes probe RL-2 was hybridized with 4 major strains of L. monocytogenes (serotype 1/2a, 1/2b, 1/2c and 4b) but not with other Listeria strains or milk spoilage bacteria. Specific FISH detection of L. monocytogenes was accomplished using the probe RL-2 when the milk contained a mixture of other bacterial species. The positive identification of L. monocytogenes in milk was completed within 5 hr (milk clearing: 40 min, hybridization: 3.5 hr, on-chip analysis: 30 min). The method presented in this study allows the specific and rapid identification of L. monocytogenes in milk.

Synergistic effect of nisin and garlic shoot juice against Listeria monocytogenes in milk

The aim of this research was to determine the synergistic effect of nisin and garlic shoot juice (GSJ) against Listeria monocytogenes ATCC 19118 found in whole (3.5%), low (1%) and skim (no fat content) milk. Garlic shoot juice (GSJ) at concentrations of 2.5%, 5% and 10% revealed strong and similar patterns of antilisterial effect against L. monocytogenes ATCC 19118 in all categories of milk. Nisin only at concentrations of 62.5, 125, 250 and 500IU/ml displayed a strong antilisterial effect as compared to the control group. Also, the synergistic combinations of GSJ (2.5%, 5%) and nisin (62.5, 125, 250 and 500IU/ml) had a remarkable antilisterial activity in all categories of whole, low and skim milk after 14 days. Results of this study indicated the synergistic effect of GSJ and nisin as a potential antilisterial agent for the food industry.

Inhibition of Listeria monocytogenes in dairy products using the bacteriocin-like peptide cerein 8A

The efficacy of the antimicrobial peptide cerein 8A to control the development of Listeria monocytogenes in milk and soft cheese was investigated. The addition of 160 AU ml − 1 cerein 8A to UHT milk resulted in a decrease of 3 log cycles in viable cells within the 14-day period at 4 °C. The viable counts of L. monocytogenes in pasteurized milk samples containing cerein 8A was lower than those observed in controls without bacteriocin. Addition of cerein 8A to Minas-type soft cheese caused a delay in the start of exponential growth phase, although similar counts were observed after day 6. When cerein 8A was used to control cheese surface contamination by L. monocytogenes, a decrease of 2 log cycles in viable counts of cerein-treated samples was observed during 30 days at 4 °C. This antimicrobial peptide shows potential use as a biopreservative for application in dairy products.

Use of mild-heat treatment following high-pressure processing to prevent recovery of pressure-injured Listeria monocytogenes in milk

This study examined the inactivation of Listeria monocytogenes in milk by high-pressure processing (HPP) and bacterial recovery during storage after HPP. We developed a technique to inhibit the bacterial recovery during storage after HPP (550 MPa for 5 min) using a mild-heat treatment (30–50 °C). Various mild-heat treatments were conducted following HPP to investigate the condition on which the bacterial recovery was prevented. Immediately after HPP of 550 MPa at 25 °C for 5 min, no L. monocytogenes cells were detected in milk regardless of the inoculum levels (3, 5, and 7 log 10 CFU/ml). However, the number of L. monocytogenes cells increased by >8 log 10 CFU/ml regardless of the inoculum levels after 28 days of storage at 4 °C. Significant recovery was observed during storage at 25 °C; the bacterial number increased by >8 log 10 CFU/ml after 3 days of storage in the case of an initial inoculum level of 7 and 5 log 10 CFU/ml. Even in the case of an initial inoculum level of 3 log 10 CFU/ml, the bacterial number reached the level of 8 log 10 CFU/ml after 7 days of storage. No bacterial recovery was observed with storage at 37 °C for 28 days. Milk samples were treated by various mild-heat treatments (30–50 °C for 5–240 min) following HPP of 550 MPa at 25 °C for 5 min, and then stored at 25 °C for 70 days. The mild-heat treatment (e.g., 37 °C for 240 min or 50 °C for 10 min) inhibited the recovery of L. monocytogenes in milk after HPP. No recovery of L. monocytogenes in milk was observed during 70-day storage at 25 °C in samples that received mild-heat treatments such as mentioned above following HPP (550 MPa for 5 min). Moreover, the mild-heat treatment conditions (temperature and holding time) required to inhibit the recovery of L. monocytogenes in milk was modelled using a logistic regression procedure. The predicted interface of recovery/no recovery can be used to calculate the mild-heat treatment condition to control bacterial recovery during storage at 25 °C after HPP (550 MPa for 5 min). The results in this study would contribute to enhance the safety of high-pressure-processed milk.

Prevalence of Listeria monocytogenes in milk, meat and foodstuff of animal origin and the phenotype of antibiotic resistance of isolated strains

In 2000–2002 samples of raw materials (milk and beef, 518 samples), ready-to-cook foods and final products from milk (30 and 200 samples) and from meat (105 and 110 samples) and swabs from surfaces in two meat-processing plants (41 samples) were examined for the presence of Listeria monocytogenes (L. monocytogenes). 70 isolates were tested using the standard microdilution method for the susceptibility to 12 antimicrobial drugs, minimum inhibitory concentration (MIC) characteristics (MIC<sub>m</sub>, MIC<sub>50</sub>, MIC<sub>90</sub>) were determined. L. monocytogenes was isolated from raw milk samples (15 samples). It was not isolated from any of the semi-finished and final milk products except for one sample of pasteurized milk. Furthermore L. monocytogenes was isolated from samples of raw beef, meat emulsion, fermented dry meat products and from swabs from production equipment. In vitro testing of susceptibility showed the considerable effectiveness of examined antimicrobial substances with streptomycin and norfloxacin being least effective (MIC<sub>m</sub> = 4 μg/ml). Apart from the intermediate resistance (MIC = 1–2 μg/ml) and resistance (MIC = 4 μg/ml) to clindamycin (37 and 5 strains, respectively), all strains were susceptible to the tested substances. While the presence of L. monocytogenes in foodstuffs, in particular, are serious, current tests of susceptibility of L. monocytogenes isolates indicate low probability untreatable infections as a result of resistant strains from foods or transfer of resistance to other microorganisms in the CzechRepublic.

Inactivation of Escherichia coli O157:H7 and Listeria monocytogenes in milk by caprylic acid and monocaprylin

Listeria monocytogenes and Escherichia coli O157:H7 are major foodborne pathogens implicated in various outbreaks involving pasteurized or unpasteurized milk, and various dairy products. The objective of this study was to determine the antibacterial effect of caprylic acid (CA, C8:0) and its monoglyceride, monocaprylin (MC) on L. monocytogenes and E. coli O157:H7 in whole milk. A five-strain mixture of E. coli O157:H7 or L. monocytogenes was inoculated in autoclaved milk (106CFU/ml) containing 0, 25, or 50mM of CA or MC. At 37°C, all the treatments, excepting 25mm CA, reduced the population of both pathogens by approximately 5.0logCFU/ml in 6h. At 24h of storage at 8°C, MC at both levels and CA at 50mM decreased L. monocytogenes and E. coli O157:H7, respectively by >5.0log CFU/ml. At 48h of 4°C storage, populations of L. monocytogenes and E. coli O157:H7 were decreased to below detection level (enrichment negative) by 50mm of MC and CA, respectively. Results indicate that MC could potentially be used to inhibit L. monocytogenes and E. coli O157:H7 in milk and dairy products, but sensory studies need to be conducted before recommending their use.

Direct detection of Listeria monocytogenes from milk by magnetic based DNA isolation and PCR

Two nucleic acid-based methods for rapid and sensitive detection of the foodborne pathogen Listeria monocytogenes in milk were developed in this study. These methods rely on paramagnetic nanoparticle-based isolation of bacterial DNA directly from milk and subsequent PCR with selective primers for the listeriolysin O (hlyA) gene. The hlyA specific product was reproducibly detected and showed a sensitivity of 10cfuml−1. The magnetic-based system had a sensitivity 10-fold higher than that of commercially available column devices. The detection limit of both methods is sufficient for direct detection of L. monocytogenes DNA in milk avoiding the enrichment culturing step, reducing the time necessary to obtain results from samples to 7h rather than the 5-day minimum required for the standard procedure. The methods developed are suitable for automation.

Rapid enumeration of Listeria monocytogenes in milk using competitive PCR

Competitive polymerase chain reaction (cPCR) was used to develop a direct enumeration method of Listeria monocytogenes in milk. Sterile milk was artificially inoculated with L. monocytogenes and DNA was extracted using guanidine thiocyanate/phenol/chloroform, followed by PCR. Several primers for L. monocytogenes hlyA gene were tested for specific detection and DG69/DG74 primer set was selected. The primer set produced a 636-bp band from L. monocytogenes, but no band appeared from the other six Listeria spp. tested. A detection limit was as few as 10 3 colony-forming unit (cfu) per 0.5 ml of milk with this primer set. When the samples were cultured at 25 °C for 15 h in a TSBY medium, even a single bacterium could be detected with this primer set by PCR. For the cPCR, hlyA gene segment was cloned in pGem-4Z vector and was modified to produce competitor DNA. The competitor DNA has the same primer binding sites and sequences as the target DNA except EcoRI site. Known amount of competitor DNA was coamplified with L. monocytogenes total DNA isolated from artificially inoculated milk. The target DNA and competitor DNA were distinguished by EcoRI digestion after cPCR. The cell number determined by cPCR was approximately equal to the colony-forming unit from conventional plate counting method. For the whole procedure, it took only 5 h.

PCR Detection of Listeria monocytogenes in Milk and Milk Products and Differentiation of Suspect Isolates

Holko I., J. Urbanova, M. Kantikova, K. Pastorova, V. KmeE: PCR Detection of Listeria monocytogenes in Milk and Milk Products and Differentiation of Suspect Isolates. Acta Vet. Brno 71, 2002: 125-131. The main goal of our work was to solve the problems of normative diagnostic method for the detection of Listeria monocytogenes in foods by STN ISO 10560 (1999), i.e. to shorten the long time of analysis (5-10 days) and differentiate the non-haemolysed strains of Listeria monocytogenes from non-pathogenic Listeria innocua yielding the same results of biochemical and serological typing (serotype 4ab). PCR was employed to confirm the method for testing 60 suspect isolates from other laboratories of the Slovak Republic. We verified the detection of Listeria monocytogenes in milk and dairy products by nested PCR. The objective of this experiment was to analyze 100 various samples by traditional cultivation method and compare the results with those yielded by PCR. The results were in good agreement; all 18 positive and 72 negative samples were detected by both methods. However, the PCR method yields results within 2 days. This makes diagnosis in food control laboratories much more efficient. For PCR detection, two pairs of primers (PRFA 1 and 2, LIP 1 and 2) were used with affinity to prfA gene involved in the regulation of listeriolysin synthesis. The size of the PCR product was 1060 bp fragment in a first step of PCR and 273 bp fragment in nested PCR. The sensitivity and reliability of PCR was comparable with conventional methods. The PCR method solved the problem of interpretation of classical biochemical and serological typing in the only one step without the necessity of using additional examinations. Furthermore, we found that in most of the strains isolated from foods with a biochemical profile of non-haemolytic Listeria monocytogenes on blood agar serotype 4ab were not Listeria monocytogenes but a non-pathogenic Listeria innocua. This finding is very important from the point of view of food evaluation according to the STN ISO 10560 (1999). Diagnostic methods, food, Listeria innocua, non-haemolysed strains of Listeria monocytogenes, prfA gene Bacteria of the genus Listeria are widely distributed in the environment and they frequently contaminate food. Listeria monocytogenes is a pathogenic species causing listeriosis, increasing incidence of which has been reported in the last two decades. Food products most frequently associated with listeriosis are soft cheeses, particularly those made from unpasteurized milk, and ready-to-eat meat-containing food products (Farber and

Inactivation of Staphylococcus Aureus and Listeria Monocytogenes in Milk and Cream of Chicken Soup by High Hydrostatic Pressure and Bacteriocins

By the application of HHP; 5.50 and 5.70 log cycle reduction was obtained for S. aureus 485, in milk and cream of chicken soup samples, respectively. Storage of HHP treated milk samples for 24 h at 4 °C gave positive result for S. aureus 765 where no growth was observed for L. monocytogenes strains. Incubation of food samples for an additional 48 h at 37 °C showed growth of L. monocytogenes strains as S. aureus strains. When a bacteriocin-based biopreservative (BP 1 ) was combined with pressurization more than 8 log-cycle reduction in cell population of the resistant strains of S. aureus and L. monocytogenes was achieved in milk. Milk and cream of chicken soup samples were stored at 25 °C and milk samples showed no growth up to 30 day where as Gram-positive bacterial strains in cream of chicken showed growth after 3 days with a rapid increase in cell number thereafter.

Detection of Listeria monocytogenes in Milk by the Polymerase Chain Reaction

A on the polymerase chain reaction (PCR) method based was developed for detection of Listeria monocytogenes in milk samples after enrichment culture. It consists of culturing samples in Listeria enrichment broth, followed by DNA extraction and detection of the organism using PCR. Dilutions of L. monocytogenes in milk were subjected to PCR amplification after enrichment culture. When determining the sensitivity of the method, it was found to be possible to detect 37 CFU (colony forming unit gl/ml) of the bacterium in milk. The method was assessed as a sensitive, specific, times-saving and practical way of detecting L. monocytogenes in milk samples.

Inactivation of Listeria monocytogenes in Milk by Pulsed Electric Field

Pasteurized whole, 2%, and skim milk were inoculated with Listeria monocytogenes Scott A and treated with high-voltage pulsed electric field (PEF). The effects of milk composition (fat content) and PEF parameters (electric field strength, treatment time, and treatment temperature) on the inactivation of the bacterium were studied. No significant differences were observed in the inactivation of L. monocytogenes Scott A in three types of milk by PEF treatment. With treatment at 25°C, 1- to 3-log reductions of L. monocytogenes were observed. PEF lethal effect was a function of field strength and treatment time. Higher field strength or longer treatment time resulted in a greater reduction of viable cells. A 4-log reduction of the bacterium was obtained by increasing the treatment temperature to 50°C. Results indicate that the use of a high-voltage PEF is a promising technology for inactivation of foodborne pathogens.

Detection and identification of Listeria monocytogenes from milk and cheese by a single-step PCR.

Primers of iap gene were used as a target to develop a PCR technique for detecting Listeria monocytogenes in milk and cheese. The PCR technique gives good results in the detection of Listeria monocytogenes either in artificially or naturally contaminated foodstuffs and has a high sensitivity and specificity. Application of this rapid diagnostic tool could provide further information about the spread of L. monocytogenes in milk and cheese.

An outbreak of gastroenteritis and fever due to Listeria monocytogenes in milk.

After an outbreak of gastroenteritis and fever among persons who attended a picnic in Illinois, chocolate milk served at the picnic was found to be contaminated with Listeria monocytogenes. In investigating this outbreak, we interviewed the people who attended the picnic about what they ate and their symptoms. Surveillance for invasive listeriosis was initiated in the states that receive milk from the implicated dairy. Stool and milk samples were cultured for L. monocytogenes. Serum samples were tested for IgG antibody to listeriolysin O. Forty-five persons had symptoms that met the case definition for illness due to L. monocytogenes, and cultures of stool from 11 persons yielded the organism. Illness in the week after the picnic was associated with the consumption of chocolate milk. The most common symptoms were diarrhea (present in 79 percent of the cases) and fever (72 percent). Four persons were hospitalized. The median incubation period for infection was 20 hours (range, 9 to 32), and persons who became ill had elevated levels of antibody to listeriolysin O. Isolates from stool specimens from patients who became ill after the picnic, from sterile sites in three additional patients identified by surveillance, from the implicated chocolate milk, and from a tank drain at the dairy were all serotype 1/2b and were indistinguishable on multilocus enzyme electrophoresis, ribotyping, and DNA macrorestriction analysis. L. monocytogenes is a cause of gastroenteritis with fever, and sporadic cases of invasive listeriosis may be due to unrecognized outbreaks caused by contaminated food.

Development of a predictive model for growth of Listeria monocytogenes in a skim milk medium and validation studies in a range of dairy products.

A predictive model based on growth of Listeria monocytogenes in milk is described. The main aim of this work was to generate a predictive model in milk acidified with lactic acid to mimic conditions found in a range of dairy products. A complete factorial design was employed to determine the effects of pH (4.5-7.5), temperature (3-35 degrees C) and salt concentration (0-8%) on growth of the organism. There were 210 design points and growth curves were individually fitted for the Gompertz function using non-linear regression. Descriptors of the curves, such as lag phase duration (LPD), exponential growth rate (EGR) and generation time (GT) were calculated and polynomial models were developed relating these to pH, temperature and salt concentration. The selected cubic polynomial model gave acceptable predictive estimates of growth and was stable, i.e. predictions were repeatable over the range of environmental variables studied. The model was further tested to determine its capacity for predicting growth of listeria in a range of dairy foods and these validation studies confirm its usefulness as a rapid means of estimating growth of the organism under specified environmental conditions.

The inhibition of Listeria monocytogenes in milk by dehydrated pediococcal spent medium

A dehydrated pediococcal spent medium (DPSM) from each Pediococcus pentosaceus L7230 and P. acidilactici Pa was used against four strains of Listeria monocytogenes (CA, OH, SA and V7). L7230 DPSM (2 mg per drop) formed zones of inhibition against all the tested Listeria strains. The growth preventing concentration (GPC) of L7230 DPSM against the strains of Listeria (10 5 cfu ml −1 ) in milk (4·5°C) was 15 mg ml −1 . The antilisterial activity of sub GPC levels of L7230 DPSM in milk (32 and 4·5°C) increased with the increase in concentration. Levels of 5 and 7·5 mg L7230 DPSM ml −1 were bacteriostatic during the first 3 days against both Listeria CA and SA. Concentrations of 5, 7·5 and 10 mg ml −1 were bactericidal against the Listeria strains after day 3, and from the start of the testing period, respectively. In many instances, the concentrations of 7·5 and 10 mg ml −1 reduced the listerial populations below 1·0 log 10 The concentration of 10 mg ml −1 had the same bactericidal effect at both 32 and 4·5°C against Listeria OH. In general, the D 7·5 values, of those Listeria strains showing biphasic curves of their populations in milk, were higher during phase I (1·9–5·4 days) than those in phase II (0·8–1·2 days). Listeria SA had the highest D 7·5 value of 5·8 days which was consistent throughout the 6 day exposure period. The control preparations (all at 10 mg ml −1 milk) used in this study such as P. acidilactici Pa DPSM, and each uninoculated lyophilized broth (ULB, pH 6·6 and 5·1) allowed L. monocytogenes strain OH to grow (32°C) to the same extent as it would in milk with no added preparation.