Isolation and characterization of nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts

Bacterial isolates from bean-sprouts were screened for anti- Listeria monocytogenes bacteriocins using a well diffusion method. Thirty-four of 72 isolates inhibited the growth of L.monocytogenes Scott A. One, HPB 1688, which had the biggest inhibition zone against L.monocytogenes Scott A, was selected for subsequent analysis. Both ribotyping and DNAsequencing of 16S ribosomal RNA gene demonstrated that the isolate was Lactococcus lactis subsp. lactis . Polymerase chain reaction and nucleotide sequencing revealed that thegenomic DNA of the bean-sprout isolates contained a nisin Z structural gene. In MRS broth,bean-sprout isolate HPB 1688 survived at 3–4·5°C for at least 20 d, grew at 4°Cand produced anti-listerial compoundsat 5°C. When co-cultured with L. monocytogenes in MRS broth, the isolate inhibited thegrowth of L. monocytogenes at 4°C after 14d and at 10°C after 2 d. When co-inoculatedwith 10²cells g⁻¹ of L.monocytogenes on fresh-cut ready-to-eat Caesar salad, L. lactis subsp. lactis (10⁸cells g⁻¹) was able to reduce the number of L. monocytogenes by 1–1·4 logs after storage for 10 d at 7° and 10°C. A bacteriocin-producing Enterococcusfaecium was also able to reduce the numbers of L. monocytogenes onCaesar salad, butdid not act synergistically when co-inoculated with L. lactis subsp. lactis .     

Survival of Listeria monocytogenes in yogurt during storage at 4°C

Cows' milk was inoculated with ca 10³and 10⁷cfu/ml Listeria monocytogenes. After fermentation at 42°C for 0–5 h, the yogurt was stored at 4°C. Low and high inocula survived for 48 h and 7 d, respectively; L. monocytogenes cells were not detectable by direct plating or cold-enrichment after 5 and 15 d, respectively. In low inoculum samples, initial pH at the time of refrigeration was 4·9; the final pH at the time of last sampling was 4·2. In the samples with high inoculum the pH decreased from 5·0 to 4·2.     

Factors affecting the growth of Listeria monocytogenes on minimally processed fresh endive

The influence of various factors on the fate of Listeria monocytogenes on cut leaves of broad-leaved endive has been studied. Factors considered were temperature, characteristics of the leaves (age, quantity and quality of the epiphytic microflora) and characteristics of the L. monocytogenes inoculum (concentration, strain). The increases in numbers of L. monocytogenes were lower than those of the aerobic mesophilic microflora at 3°, 6°, 10° and 20°C. Doubling times of the populations of L. monocytogenes were in the same order of magnitude as those of aerobic bacteria at 10° and 20°C, but longer at 3° and 6°C. There were positive significant correlations between growth of L. monocytogenes and populations of aerobic bacteria, and between growth of L. monocytogenes and extent of spoilage on the leaves.
Of 225 bacteria isolated from the leaves, 84% were identified as fluorescent pseudomonads; there was no difference in the species isolated from leaves that showed a low growth of L. monocytogenes and leaves that showed a high growth of L. monocytogenes. Populations of L. monocytogenes increased faster during the first 2 and 4 d of storage at 10°C on leaves inoculated with 10–10³ cfu g^-1 than on leaves inoculated with about 10⁵ cfu g^-1, but the population reached after 7 d was lower. The behaviour of L. monocytogenes was similar among the three strains tested.     

Fate of low temperature and acid-adapted Yersinia enterocolitica and Listeria monocytogenes that contaminate lactic acid decontaminated meat during chill storage

Pathogens found in the environment of abattoirs may become adapted to lactic acid used to decontaminate meat. Such organisms are more acid tolerant than non-adapted parents and can contaminate meat after lactic acid decontamination (LAD). The fate of acid-adapted Yersinia enterocolitica and Listeria monocytogenes, inoculated on skin surface of pork bellies 2 h after LAD, was examined during chilled storage. LAD included dipping in 1%, 2% or 5% lactic acid solutions at 55°C for 120 s. LAD brought about sharp reductions in meat surface pH, but these recovered with time after LAD at ≈1–1·5 pH units below that of water-treated controls. Growth permitting pH at 4·8–5·2 was reached after 1% LAD in less than 0·5 d (pH 4·8–5·0), 2% LAD within 1·5 d (pH 4·9–5·1) and after 5% LAD (pH 5·0–5·2) within 4 d. During the lag on 2% LAD meat Y. enterocolitica counts decreased by 0·9 log₁₀ cfu per cm² and on 5% LAD the reduction was more than 1·4 log₁₀ cfu per cm². The reductions in L. monocytogenes were about a third of those in Y. enterocolitica . On 1% LAD the counts of both pathogens did not decrease significantly. The generation times of Y. enterocolitica and L. monocytogenes on 2–5% LAD meats were by up to twofold longer than on water-treated controls and on 1% LAD-treated meat they were similar to those on water-treated controls. Low temperature and acid-adapted L. monocytogenes and Y. enterocolitica that contaminate skin surface after hot 2–5% LAD did not cause an increased health hazard, although the number of Gram-negative spoilage organisms were drastically reduced by hot 2–5% LAD and intrinsic (lactic acid content, pH) conditions were created that may benefit the survival and the growth of acid-adapted organisms.     

Comparison of the growth of Listeria monocytogenes in unirradiated and irradiated cook-chill roast beef and gravy at refrigeration temperatures

Specific growth rates of two strains of Listeria monocytogenes in unirradiated and irradiated (2 kGy) roast beef and gravy stored at 5° and 10°C were found to be similar. However, exponential growth of L. monocytogenes after irradiation was preceded by an extended lag period of 6–9 d at 5°C and 3–4 d at 10°C, compared with lag periods of 1–2 d and <0.1 d in unirradiated beef and gravy stored similarly.     

Inhibition of Listeria monocytogenes by enterocin 4 during the manufacture andripening of Manchego cheese

The inhibitory effect of enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4, on Listeria monocytogenes strains Ohio and Scott A during themanufacture and ripening of Manchego cheese was investigated. Raw ewe's milk wasinoculated with ca 10⁵ cfu ml⁻¹ of L.monocytogenes and with 1% of a commercial lactic starter, 1% of an Ent. faecalis INIA 4 culture, or 1% of each culture. Manchego cheeses were manufactured according tousual procedures. Listeria monocytogenes Ohio counts decreased by 3 log units after8 h and by 6 log units after 7 d in cheese made from milk inoculated with Ent. faecalis INIA 4 or with both cultures, whereas no inhibition was recorded after 60 d in cheese made frommilk inoculated with commercial lactic starter. Listeria monocytogenes Scott A wasnot inhibited by enterocin 4 during cheese manufacture, but decreases of 1 log unit after 7 d andof 2 log units after 60 d were achieved in cheese made from milk inoculated with bothcommercial lactic starter and Ent. faecalis INIA 4.     

The effect of nisin on Listeria monocytogenes in culture medium and long-life cottage cheese

M.A.S.S. FERREIRA AND B.M. LUND. 1996. The sensitivity to nisin of 27 strains of Listeria monocytogenes , four of L. innocua and one of L. ivanovii was estimated at pH 6.8 and pH 5.5. Strains of L. monocytogenes showed differences in sensitivity which were not correlated with serotype. Strains of L. innocua were as resistant as the most resistant strains of L. monocytogenes , whereas the strain of L. ivanovii was relatively sensitive. Two of the most resistant strains of L. monocytogenes multiplied in aerated liquid medium adjusted to pH 5.0 with HCl, incubated at 20°C; nisin, 500 IU ml^-1, prevented multiplication and caused death. Following inoculation of a resistant strain into long-life cottage cheese, pH 4.6–4.7, the number of viable L. monocytogenes decreased approximately 10-fold during storage at 20°C for 7 d; addition of nisin, 2000 IU g^-1, to the cottage cheese increased the rate of inactivation to approximately a 1000-fold decrease in 3 d.     

The use of the bacteriocin, nisin, as a preservative in ricotta-type cheeses to control the food-borne pathogen Listeria monocytogenes

The efficacy of nisin to control the food-borne pathogen Listeria monocytogenes in ricotta-type cheeses over long storage (70 d) at 6–8°C was determined. Cheeses were prepared from unpasteurized milk by direct acidification with acetic acid (final pH 5·9) and/or calcium chloride addition during heat treatment. Nisin was added in the commercial form of Nisaplin^® pre-production to the milk. Each batch of cheese was inoculated with 10²–10³ cfu g⁻¹ of a five-strain cocktail of L. monocytogenes before storage. Shelf-life analysis demonstrated that incorporation of nisin at a level of 2·5 mg l⁻¹ could effectively inhibit the growth of L. monocytogenes for a period of 8 weeks or more (dependent on cheese type). Cheese made without the addition of nisin contained unsafe levels of the organism within 1–2 weeks of incubation. Measurement of initial and residual nisin indicated a high level of retention over the 10-week incubation period at 6–8°C, with only 10–32% nisin loss.     

The effect of slurry storage and anaerobic digestion on survival of pathogenic bacteria

The decline in viable numbers of Salmonella typhimurium, Yersinia enterocolitica and Listeria monocytogenes in beef cattle slurry is temperature-dependent; they decline more rapidly at 17°C than at 4°C. Mesophilic anaerobic digestion caused an initial rapid decline in the viable numbers of Escherichia coli, Salm. typhimurium, Y. enterocolitica and L. monocytogenes. This was followed by a period in which the viable numbers were not reduced by 90%. The T₉₀ values of E. coli, Salm. typhimurium and Y. enterocolitica ranged from 0.7 to 0.9 d during batch digestion and 1.1 to 2.5 d during semi-continuous digestion. Listeria monocytogenes had a significantly higher mean T₉₀ value during semi-continuous digestion (35.7 d) than batch digestion (12.3 d). Anaerobic digestion had little effect in reducing the viable numbers of Campylobacter jejuni.     

Effects of gaseous environment and temperature on the storage behaviour of Listeria monocytogenes on chicken breast meat

Portions of skinless chicken breast meat (pH 5·8) were inoculated with a strain of Listeria monocytogenes and stored at 1, 6 or 15°C in (1) aerobic conditions; (2) 30% CO₂+ air; (3) 30% CO₂+ N₂; and (4) 100% CO₂. When samples were held at 1°C the organism failed to grow under any of the test conditions, despite marked differences between treatments in spoilage rate and ultimate microflora. At 6°C counts of L. monocytogenes increased ca 10-fold in aerobic conditions before spoilage of the meat, but only when the inoculum culture was incubated at 1°C rather than 37°C. In CO₂ atmospheres growth of L. monocytogenes was inhibited on meat held at 6°C, especially under 100% CO₂. By contrast, storage at 15°C led to spoilage of the meat within 2 d, in all gaseous environments, and listeria levels increased up to 100-fold. Differences in the behaviour of L. monocytogenes on poultry and red meats are discussed.     

Survival of Listeria monocytogenes in sea water and effect of exposure on thermal resistance

Survival, recoverability and sublethal injury of two strains of Listeria monocytogenes , Scott A and an environmental strain KM, on exposure to sea water at 12·8 or 20·8 °C was determined using in situ diffusion chambers. Plate counts were used to assess recoverability and injury while 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction was used to determine respiratory activity. T₉₀ values (times for 10-fold decreases in numbers of recoverable cells) on non-selective medium (trypticase soya agar with 0·6% yeast extract) at 12·8 and 20·8 °C were 61·7 and 69·2 h for L. monocytogenes Scott A, and 103·0 and 67·0 h for L. monocytogenes KM, respectively. On selective medium (Oxford agar), T₉₀ values at 12·8 and 20·8 °C were 60·6 and 56·9 h for L. monocytogenes Scott A, and 83·0 and 65·9 h for L. monocytogenes KM, respectively. With Scott A, the percentage of sublethally injured cells at 12·8 and 20·8 °C was 1·7 and 17·7%, respectively, while for KM the values were 19·0 and 1·6%, respectively. The fraction of cells reducing CTC but which were not recoverable on plating progressively increased on exposure to sea water. Listeria monocytogenes KM challenged at 58 °C showed an apparent increase in heat resistance after exposure to sea water at 20·8 °C for 7 d ( D ₅₈= 2·64 min) compared with before exposure ( D ₅₈= 1·24). This increase in thermal resistance was not apparent at temperatures greater than 63 °C, and analysis of the best-fit regression lines fitted to the thermal data obtained from the two cell populations indicated that their thermal resistance was not significantly different ( P > 0·05) over the temperature range tested (58–62 °C).     

Influence of strain and trisodium phosphate concentration on growth parameters of Listeria monocytogenes in vitro

AIMS: The present study was conducted to determine the influence of strain and trisodium phosphate (TSP) concentration in the growth of Listeria monocytogenes in vitro. METHODS AND RESULTS: Three strains (ATCC 11916, 64d, isolated from chicken meat, and M2-5b, a clinical animal isolate) were inoculated in broth with 0, 0.5, 1 and 1.5% (w/v) of TSP. The shortest lag phase and highest maximum rate of growth (mu) were obtained in the presence of 0.5% TSP. In contrast, the highest lag phase and lowest mu were obtained with 1.5% TSP. For each TSP concentration, significant differences (P < 0.05) in lag phase and mu of the three L. monocytogenes strains were observed. CONCLUSION: The behaviour of L. monocytogenes is significantly influenced by both the origin of the strain and the salt concentration. Significance and Impact of the Study: Our results indicate the importance of choosing an adequate TSP concentration for the decontamination of foods, because low concentrations could favour the growth of L. monocytogenes.     

Growth of Listeria monocytogenes at different pH values in uncultured whey or whey cultured with Penicillium camemberti

Wheys from making Camembert cheese were either uncultured or cultured with Penicillium camemberti, adjusted to pH 5.0, 5.2, 5.4, 5.6, 6.2, and 6.8, and filter sterilized. Whey samples were inoculated to contain 100 to 500 Listeria monocytogenes (strains Scott A, V7, CA, or OH) cfu/mL and incubated at 6 degrees C. Counts of L. monocytogenes were obtained by surface plating appropriate dilutions on Tryptose Agar. Listeria monocytogenes failed to grow at or below pH 5.4; except for strains Scott A and OH which grew in cultured whey at pH 5.4 and attained populations of 7.8 x 10(3) and 5.4 x 10(4) cfu/mL, respectively, after 35 d of storage. In uncultured whey at pH 5.6, 6.2, and 6.8, populations of L. monocytogenes increased from 7.20 to 7.81, 7.51 to 8.23, and 7.48 to 8.08 log10 cfu/mL, respectively, after 35 d of storage at 6 degrees C. In cultured whey at pH 5.6, 6.2, and 6.8, numbers of L. monocytogenes increased from 7.53 to 8.13, 7.82 to 8.55, and 7.95 to 8.80 log10 cfu/mL, respectively, after 35 d of storage. Generation times for L. monocytogenes at 6 degrees C in uncultured whey at pH 5.6, 6.2, and 6.8 ranged between 25.3 and 31.6 h, 14.8 and 21.1 h, and 14.0 and 19.4 h, respectively, depending on the Listeria strain. In contrast, generation times were significantly (p less than 0.05) shorter in cultured whey and ranged between 16.6 and 27.4 h, 10.3 and 16.6 h, and 17.4 and 16.3 h at pH values of 5.6, 6.2, and 6.8, respectively.     

Control of Listeria monocytogenes in a biofilm by competitive-exclusion microorganisms

Biofilms from drains in food processing facilities with a recent history of no detectable Listeria monocytogenes in floor drains were cultured for microorganisms producing antilisterial metabolites. A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37 degrees C for activities that were bactericidal or inhibitory to L. monocytogenes, by two agar plate assays. Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity. All 24 isolates which produced metabolites inhibitory to L. monocytogenes were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE). A five-strain mixture of 10(3) CFU of L. monocytogenes/ml and 10(5) CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37 degrees C for 24 h, 15 degrees C for 14 days, 8 degrees C for 21 days, and 4 degrees C for 28 days. Substantial inhibition of L. monocytogenes growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37 degrees C, two at 15 and 8 degrees C, and three at 4 degrees C. The inhibitory isolates were identified as Enterococcus durans (six isolates), Lactococcus lactis subsp. lactis (two isolates), and Lactobacillus plantarum (one isolate). The anti-L. monocytogenes activity of these isolates was evaluated in biofilms of L. monocytogenes on stainless steel coupons at 37, 15, 8, and 4 degrees C. Results revealed that two isolates (E. durans strain 152 and L. lactis subsp. lactis strain C-1-92) were highly inhibitory to L. monocytogenes (growth inhibition of >5 log(10) CFU of L. monocytogenes/cm(2)). These two bacterial isolates appear to be excellent competitive-exclusion candidates to control L. monocytogenes in biofilms at environmental temperatures of 4 to 37 degrees C.     

Effects of essential oil from mint (Mentha piperita) on Salmonella enteritidis and Listeria monocytogenes in model food systems at 4° and 10°C

The effect of mint ( Mentha piperita ) essential oil (0·5, 1·0, 1·5 and 2·0%, v/w) on Salmonella enteritidis and Listeria monocytogenes in a culture medium and three model foods; tzatziki (pH 4·5), taramosalata (pH 5·0) and pâté (pH 6·8), inoculated at 10⁷ cfu g^-1, at 4° and 10°C for ca 1 week was studied. In the culture medium supplemented with the essential oil, no growth was observed over 2 d at 30°C determined by a conductance method with a Malthus 2000 growth analyser. Salmonella enteritidis died in tzatziki in all treatments and declined in the other foods except for pâté at 10°C as judged with viable counts. Listeria monocytogenes populations showed a declining trend towards the end of the storage period but was increased in pâté. Mint essential oil antibacterial action depended mainly on its concentration, food pH, composition, storage temperature and the nature of the micro-organism.     

Pulsed-field fingerprinting of listeriae: identification of genomic divisions for Listeria monocytogenes and their correlation with serovar.

Clamped homogeneous electric field (CHEF) electrophoresis was optimized for genomic analyses of Listeria monocytogenes. Various human, animal, food, and environmental isolates, as well as strains representing other Listeria species, were separately digested with rarely cutting endonucleases. Of 176 L. monocytogenes strains analyzed, the enzymes AscI and ApaI established 63 and 72 unique restriction endonuclease digestion profiles (REDP), respectively. The 22 non-L. monocytogenes strains exhibited 18 AscI and 19 ApaI unique REDP. Statistical analyses of REDP information using the Dice coincidence index and principal component analysis revealed two distinct genomic divisions of L. monocytogenes that also correlated with the flagellar (H) antigen type: division I contained serovar 1/2a, 1/2c, 3a, and 3c stains and division II contained serovar 1/2b, 3b, 4b, 4d, and 4e strains. Division I isolates digested with ApaI were further grouped into cluster IA (serovar 1/2c and 3c) and cluster IB (serovar 1/2a and 3a) strains. Likewise, division II isolates digested with ApaI were further grouped into cluster IIA (serovar 1/2b and 3b) and cluster IIB (serovar 4b, 4d, and 4e) strains. These data indicate that genotypic data generated by CHEF can be directly related to phenotypic data generated by serotyping for establishing the overall relatedness of isolates. Moreover, these data further substantiate that CHEF analysis is a reproducible and highly discriminating method for characterizing L. monocytogenes strains at the molecular level.     

Shelf life and safety aspects of chilled cooked and peeled shrimps (Pandalus borealis) in modified atmosphere packaging

AIMS: To evaluate the growth of Listeria monocytogenes and shelf life of cooked and peeled shrimps in modified atmosphere packaging (MAP). METHODS AND RESULTS: Storage trials with naturally contaminated cooked and peeled MAP shrimps (Pandalus borealis) were carried out at 2, 5 and 8 degrees C. Challenge tests at the same conditions were performed after inoculation with Listeria monocytogenes. Both storage trials and challenge tests were repeated after 4 months of frozen storage (-22 degrees C). Brochothrix thermosphacta and Carnobacterium maltaromaticum were responsible for sensory spoilage of cooked and peeled MAP shrimps. In challenge tests, growth of L. monocytogenes was observed at all of the storage temperatures studied. At 5 and 8 degrees C the concentration of L. monocytogenes increased more than a 1000-fold before the product became sensory spoiled whereas this was not observed at 2 degrees C. Frozen storage had only a minor inhibiting effect on growth of L. monocytogenes in the thawed product. CONCLUSIONS: To prevent L. monocytogenes becoming a safety problem, cooked and peeled MAP shrimps should be distributed at 2 degrees C and with a maximum shelf life of 20-21 d. At higher temperatures shelf life is significantly reduced. SIGNIFICANCE AND IMPACT OF THE STUDY: Information is provided to establish shelf life of cooked and peeled MAP shrimps.     

Effect of rising temperature on the heat resistance of Listeria monocytogenes in meat emulsion

The heat resistance of a strain of L. monocytogenes was determined both in broth and in meat emulsion. The D -values for meat emulsion were approximately two to three times higher than those for broth and also the z -value increased significantly. The micro-organism proved to be more resistant when the cells were heated up slowly (0·5°C/min) to constant temperatures of 60, 63 and 66°C in meat emulsion. The D ₆₀, D ₆₃ and D ₆₆ were, respectively 12·95, 5·4 and 2·3 min. Results may have implications in the survival of Listeria monocytogenes in particular food preparations.     

Simple method to observe the adaptive response of Listeria monocytogenes in food

A simple, novel method for determining stress-adaptive response of Listeria monocytogenes in food systems is presented. The method involves plating samples on Listeria-selective agar (LSA) acidified to pH 5.25 with incubation at 36 degrees C for 60 h to detect acid adaptation and plating on LSA with 70 gl-1 NaCl and incubation at 7 degrees C for 7 d to detect cold-osmotic adaptation. Adapted cells produced larger colonies (> 1 mm) under these conditions than unadapted cells. Scot A (97%) and Brie-1 (100%) cells incubated in milk at pH 5 for 3 h manifested the acid-adapted colony type compared with 6% and 21% of viable cells in the unstressed control population. After a 5-d adaptation period at 4 degrees C in milk with 80 gl-1 salt, 29% of Scot A and 91% of Brie-1 viable cells exhibited the adapted colony type compared with < 1% of the unstressed control population. Stress-adapted L. monocytogenes were isolated from soft cheese held for 42 d at 10 C.     

Growth of Listeria monocytogenes at refrigeration temperatures

The growth of three strains of Listeria monocytogenes at refrigeration temperatures (-0.5 to 9.3°C) in chicken broth and/or UHT milk was determined using a rocking temperature gradient incubator. Minimum growth temperatures ranged from -0.1 to -0.4°C for the three strains. Lag times of 1–3 d and 3 to >34 d were observed with incubation at 5 and 0°C respectively. Corresponding generation times ranged from 13–24 h at 5°C and 62–131 h at 0°C. The type of culture medium had an influence on both the rate and extent of growth. Incubation of cultures at 4°C before inoculation caused a marked reduction in the lag time when compared with cultures which had been previously incubated at 30°C.