Inactivation of Listeria monocytogenes in Skim Milk and Liquid Egg White by Antimicrobial Bottle Coating with Polylactic Acid and Nisin

ABSTRACT: This study was to develop an antimicrobial bottle coating method to reduce the risk of outbreaks of human listeriosis caused by contaminated liquid foods. Liquid egg white and skim milk were inoculated with Listeria monocytogenes Scott A and stored in glass jars that were coated with a mixture of polylactic acid (PLA) polymer and nisin. The efficacy of PLA per nisin coating in inactivating L. monocytogenes was investigated at 10 and 4 °C. The pathogen grew well in skim milk without PLA/nisin coating treatments, reaching 8 log CFU/mL after 10 d and then remained constant up to 42 d at 10 °C. The growth of Listeria at 4 °C was slower than that at 10 °C, taking 21 d to obtain 8 log CFU/mL. At both storage temperatures, the PLA coating with 250 mg nisin completely inactivated the cells of L. monocytogenes after 3 d and throughout the 42-d storage period. In liquid egg white, Listeria cells in control and PLA coating without nisin samples declined 1 log CFU/mL during the first 6 d at 10 °C and during 28 d at 4 °C, and then increased to 8 or 5.5 log CFU/mL. The treatment of PLA coating with 250 mg nisin rapidly reduced the cell numbers of Listeria in liquid egg white to undetectable levels after 1 d, then remained undetectable throughout the 48 d storage period at 10 °C and the 70 d storage period at 4 °C. These data suggested that the PLA/nisin coating treatments effectively inactivated the cells of L. monocytogenes in liquid egg white and skim milk samples at both 10 and 4 °C. This study demonstrated the commercial potential of applying the antimicrobial bottle coating method to milk, liquid eggs, and possibly other fluid products.     

The inactivation of Listeria monocytogenes by pulsed electric field (PEF) treatment in a static chamber

An experimental analysis of the effect of pulsed electric field (PEF) energy on the inactivation of Listeria monocytogenes was conducted using a custom-designed static chamber and a gel suspension medium for treatment. This allowed PEF energy to be delivered to the suspension under near isothermal conditions. The effects of variations in the number of pulses (5–50 pulses), electric field strength (15–30 kV/cm), temperature (0–60°C) and media bases (water and skim milk) on the inactivation of L. monocytogenes were examined. At temperatures less than 50°C a maximum of 1 log reduction was obtained for L. monocytogenes regardless of pulse number or electric field strength within the ranges examined. In skim milk no reduction occurred. At 50°C and 55°C synergy between PEF and thermal energy was observed. The experimental approach separated the contribution of PEF and thermal energy to total kill and thus allowed this synergy to be quantified. At 55°C the kill due to PEF energy increased to 4.5 logs with another 4.5 logs reduction attributable to thermal energy. It appears that under the conditions of this study PEF alone has a very limited effect on the reduction of L. monocytogenes. However, the addition of thermal energy not only contributed to the kill, but also increased the susceptibility of L. monocytogenes to PEF energy.     

Application of bacteriocinogenic Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch in the control of Listeria monocytogenes in fresh Minas cheese

Several strains of Enterococcus spp. are capable of producing bacteriocins with antimicrobial activity against important bacterial pathogens in dairy products. In this study, the bacteriocins produced by two Enterococcus strains (Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch), isolated from cheeses, were characterized and tested for their capability to control growth of Listeria monocytogenes 426 in experimentally contaminated fresh Minas cheese during refrigerated storage. Both strains were active against a variety of pathogenic and non-pathogenic microorganisms and bacteriocin absorption to various L. monocytogenes, Enterococcus faecalis ATCC 19443 and Lactobacillus sakei ATCC 15521 varied according to the strain and the testing conditions (pH, temperature, presence of salts and surfactants). Growth of L. monocytogenes 426 was inhibited in cheeses containing E. mundtii CRL35 up to 12 days at 8 °C, evidencing a bacteriostatic effect. E. faecium ST88Ch was less effective, as the bacteriostatic affect occurred only after 6 days at 8 °C. In cheeses containing nisin (12.5 mg/kg), less than one log reduction was observed. This research underlines the potential application of E. mundtii CRL35 in the control of L. monocytogenes in Minas cheese.     

Bactericidal activity of lauric arginate in milk and Queso Fresco cheese against Listeria monocytogenes cold growth

Lauric arginate (LAE) at concentrations of 200 ppm and 800 ppm was evaluated for its effectiveness in reducing cold growth of Listeria monocytogenes in whole milk, skim milk, and Queso Fresco cheese (QFC) at 4°C for 15 to 28 d. Use of 200 ppm of LAE reduced 4 log cfu/mL of L. monocytogenes to a nondetectable level within 30 min at 4°C in tryptic soy broth. In contrast, when 4 log cfu/mL of L. monocytogenes was inoculated in whole milk or skim milk, the reduction of L. monocytogenes was approximately 1 log cfu/mL after 24 h with 200 ppm of LAE. When 800 ppm of LAE was added to whole or skim milk, the initial 4 log cfu/mL of L. monocytogenes was nondetectable following 24 h, and no growth of L. monocytogenes was observed for 15 d at 4°C. With surface treatment of 200 or 800 ppm of LAE on vacuum-packaged QFC, the reductions of L. monocytogenes within 24 h at 4°C were 1.2 and 3.0 log cfu/g, respectively. In addition, the overall growth of L. monocytogenes in QFC was decreased by 0.3 to 2.6 and by 2.3 to 5.0 log cfu/g with 200 and 800 ppm of LAE, respectively, compared with untreated controls over 28 d at 4°C. Sensory tests revealed that consumers could not determine a difference between QFC samples that were treated with 0 and 200 ppm of LAE, the FDA-approved level of LAE use in foods. In addition, no differences existed between treatments with respect to flavor, texture, and overall acceptability of the QFC. Lauric arginate shows promise for potential use in QFC because it exerts initial bactericidal activity against L. monocytogenes at 4°C without affecting sensory quality.     

Antimicrobial Activity of Vanillin and Mixtures with Cinnamon and Clove Essential Oils in Controlling <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 in Milk

The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of vanillin against Listeria monocytogenes Scott A and Escherichia coli O157:H7 was determined in tripticase soy broth (TSB), pH 7 and 6, incubated at 35 °C/24 h and in semi-skim milk incubated at 35 °C/24 h and 7 °C/14 days. The influence of the fat content of milk on the antimicrobial activity of vanillin was tested in whole and skim milk incubated at 7 °C/14 days. Mixtures of clove and cinnamon with vanillin were also evaluated in semi skim milk incubated at 7 °C. The MICs for L. monocytogenes were 3,000 ppm in TSB (pH 7) and 2,800 ppm in TSB (pH 6). The MICs for E. coli O157:H7 were 2,800 ppm in TSB (pH 7) and 2,400 ppm in TSB (pH 6). The MBCs in TSB were 8,000 ppm for L. monocytogenes and 6,000 ppm for E. coli O157:H7. The pH values assayed did not influence significantly the MIC or MBC in TSB. The MICs in semi-skim milk for L. monocytogenes and E. coli O157:H7 were 4,000 and 3,000 ppm at 35 °C/24 h, and 2,500 and 1,000 ppm at 7 °C/7 days, respectively. The MBCs were 20,000 ppm for L. monocytogenes and 11,000 ppm for E. coli O157:H7. High incubation temperatures did not affect the MBC but increased the MIC of the vanillin in milk. This effect could be attributed to the increased membrane fluidity and to the membrane perturbing activity of vanillin at low temperatures. The fat in milk reduced significantly the antimicrobial activity of vanillin, probably due to effect protective of the fat molecules. Mixtures of clove and cinnamon leaves inhibited the growth of L. monocytogenes in a similar way that vanillin alone but had a synergistic effect on the E. coli O157:H7. Mixtures of cinnamon bark and vanillin had always a synergistic effect and some of the combination assayed showed bactericidal activity on the population of L. monocytogenes and E. coli O 157:H7.     

Partial Characterization of Nine Bacteriocins Produced by Lactic Acid Bacteria Isolated from Cold-Smoked Salmon with Activity against Listeria monocytogenes

Nine LAB bacteriocin-producers, isolated from vacuum-packaged cold-smoked salmon (CSS), were phenotypically and genotypically identified as Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus fermentum, Enterococcus faecium, and Pediococcus acidilactici. Their bacteriocins were partially characterized. The antimicrobial spectrum was determined against Listeria monocytogenes, E. faecalis, E. faecium, and Staphylococcus aureus. The molecular size of bacteriocins ranged from 2.8 to 4.5 kDa. They were inactivated by treatment with proteolytic enzymes but not by lipolytic or glycolytic enzymes. Maximal activity against L. monocytogenes ranged between 800 and 10000 AU/mL at pH 6.5. Most of the bacteriocins maintained full activity in a pH range of 2.0 to 8.0 but were partially or completely inactivated at pH 10.0. After heating at 60°C and 100°C, only two bacteriocins from Lb. curvatus strains partially lost activity. All bacteriocins showed a narrow spectrum of activity and a high anti-listerial activity, which is characteristic of the class IIa bacteriocins. Isolated bacteriocin-producing LAB could be used successfully in the bio-preservation of CSS and development of new potential bio-preservatives for CSS active against L. monocytogenes.     

Effect of heat shock on thermal tolerance and susceptibility of Listeria monocytogenes to other environmental stresses

In the present study, Listeria monocytogenes Scott A, V7 and CCRC 14930 were first subjected to heat shock at 45°C for 1 h or at 48°C for 10 min. Thermal tolerance at 55°C and survival of the heat shocked as well as non-heat shocked cells of L. monocytogenes in the presence of 25% NaCl, 0.01% crystal violet, 0.1% H₂O₂, and 18% ethanol were examined.It was found that heat shock response of L. monocytogenes varied with strains, the condition of heat shock treatment and type of subsequent stress. Compared with the non-heat shocked cells, the 45°C-1 h heat shocked cells of strains Scott A and V7 showed an increased survival after exposure to 55°C for 60 min. Meanwhile, survival of the 48°C-10 min heat shocked L. monocytogenes cells, regardless of strain, exhibited no significant difference (p>0.05) with their respective control cells. Generally, heat shocking at 45°C for 1 h increased the tolerance of L. monocytogenes to NaCl, ethanol and crystal violet. On the other hand, heat shocking at 48°C for 10 min although increased the resistance of L. monocytogenes to NaCl reduced its resistance to H₂O₂ and crystal violet.     

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.     

Lactose oxidase: An enzymatic approach to inhibit Listeria monocytogenes in milk

Listeria monocytogenes is a ubiquitous pathogen that can cause morbidity and mortality in immunocompromised individuals. Growth of L. monocytogenes is possible at refrigeration temperatures due to its psychrotrophic nature. The use of antimicrobials in dairy products is a potential way to control L. monocytogenes growth in processes with no thermal kill step, thereby enhancing the safety of such products. Microbial-based enzymes offer a clean-label approach for control of L. monocytogenes outgrowth. Lactose oxidase (LO) is a microbial-derived enzyme with antimicrobial properties. It oxidizes lactose into lactobionic acid and reduces oxygen, generating H₂O₂. This study investigated the effects of LO in UHT skim milk using different L. monocytogenes contamination scenarios. These LO treatments were then applied to raw milk with various modifications; higher levels of LO as well as supplementation with thiocyanate were added to activate the lactoperoxidase system, a natural antimicrobial system present in milk. In UHT skim milk, concentrations of 0.0060, 0.012, and 0.12 g/L LO each reduced L. monocytogenes counts to below the limit of detection between 14 and 21 d of refrigerated storage, dependent on the concentration of LO. In the 48-h trials in UHT skim milk, LO treatments were effective in a concentration-dependent fashion. The highest concentration of LO in the 21-d trials, 0.12 g/L, did not show great inhibition over 48 h, so concentrations were increased for these experiments. In the lower inoculum, after 48 h, a 12 g/L LO treatment reached levels of 1.7 log cfu/mL, a reduction of 1.3 log cfu/mL from the initial inoculum, whereas the control grew out to approximately 4 log cfu/mL, an increase of 1 log cfu/mL from the inoculum on d 0. When a higher challenge inoculum of 5 log cfu/mL was used, the 0.12 g/L and 1.2 g/L treatments reduced the levels by 0.2 to 0.3 log cfu/mL below the initial inoculum and the 12 g/L treatment by >1 log cfu/mL below the initial inoculum by hour 48 of storage at refrigeration temperatures. After the efficacy of LO was determined in UHT skim milk, LO treatments were applied to raw milk. Concentrations of LO were increased, and the addition of thiocyanate was investigated to supplement the effect of the lactoperoxidase system against L. monocytogenes. When raw milk was inoculated with 2 log cfu/mL, 1.2 g/L LO alone and combined with sodium thiocyanate reduced ~0.8 log cfu/mL from the initial inoculum on d 7 of storage, whereas the control grew out to >1 log cfu/mL from the initial inoculum. Furthermore, in the higher inoculum, 1.2 g/L LO combined with sodium thiocyanate reduced L. monocytogenes counts from the initial inoculum by >1 log cfu/mL, whereas the control grew out 2 log cfu/mL from the initial inoculum. Results from this study suggest that LO is inhibitory against L. monocytogenes in UHT skim milk and in raw milk. Therefore, LO may be an effective treatment to prevent L. monocytogenes outgrowth, increase the safety of raw milk, and be used as an effective agent to prevent L. monocytogenes proliferation in fresh cheese and other dairy products. This enzymatic approach is a novel application to control the foodborne pathogen L. monocytogenes in dairy products.     

Development of a loop-mediated isothermal amplification assay for detecting Listeria monocytogenes prfA in milk

A rapid and sensitive loop-mediated isothermal amplification (LAMP) assay was developed for detecting Listeria monocytogenes prfA in milk. The inclusivity of 23 L. monocytogenes and the exclusivity of 16 non-L. monocytogenes strains were both 100% in the assay. The limit of detection (LoD) of the LAMP assay in Listeria enrichment broth (LEB) was 2.22 CFU/mL after 12 h and 24 h of incubation. The LoDs of the LAMP assay in LEB with artificially contaminated milk (LEB-M) incubated for 12 h (2.22×10¹ CFU/mL) and 24 h (2.22 CFU/mL) were lower than those of the PCR and real-time PCR assays. Comparison of the LoDs in LEB with those in LEB-M showed that the LAMP assay was less influenced by the milk compounds than the real-time PCR assay. Our results indicate that the LAMP assay can be utilized as a potential screening tool for L. monocytogenes in milk.     

Characterization of cottage cheese using Weissella cibaria D30: Physicochemical,antioxidant, and antilisterial properties

This study aimed to evaluate the potential of Weissella cibaria D30 as an adjunct culture in cottage cheese, including an assessment of antioxidant, antilisterial, and compositional parameters. Cottage cheese samples were manufactured using a commercial starter culture and probiotic strains Lactobacillus rhamnosus GG (GG) or W. cibaria D30 (W) and without probiotic (control). Samples were stored at 4 ± 1°C for 28 d. Bacterial cell counts (log cfu/g) of control, GG, and W samples were counted at 0, 7, 14, 21, and 28 d. Counts of W. cibaria D30 in the W samples remained at 6.85 log cfu/g after 28 d. Total solids, fat, protein, ash, and pH were measured and no significant differences were observed in compositional parameters or pH after 28 d of storage in all cheeses except those inoculated to Listeria monocytogenes. To measure the antilisterial effect, Listeria monocytogenes was inoculated into the cottage cheese samples and bacterial cell counts were obtained at 0, 6, 12, 24, 48, 72, 96, 120, and 144 h. Listeria monocytogenes counts were less than the analytical limit of detection (<10 cfu/g) in the inoculated GG and W samples, whereas the counts of L. monocytogenes in the inoculated control sample remained at 3.0 log cfu/g after 144 h. We used the DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging activity assays to assess antioxidant activity: GG and W samples exhibited significant increases in antioxidant activity compared with the control sample. These results indicate that W. cibaria D30 has potential as an adjunct culture in the dairy industry.     

Potential production and preservation of dahi by Lactococcus lactis W8, a nisin-producing strain

Lactococcus lactis W8 produced nisin concomitantly while fermenting milk to “dahi”, a traditional Indian fermented milk. The activity of nisin was detected at 3 h of fermentation, which increased in parallel to growth of the organism and reached its maximum at 6 h. The activity remained essentially stable thereafter. At 7 h of fermentation of milk with the strain L. lactis W8 the pH of the medium dropped to 4.2, when the milk became converted to dahi. The produced dahi displayed antibacterial property against spoilage and pathogenic bacteria including Listeria monocytogenes. When L. monocytogenes was mixed with dahi at 5.2 log CFU/ml and stored at 4 °C, the number of L. monocytogenes gradually decreased and became undetectable at 10 h. L. lactis W8 appeared to be a suitable starter culture for production of dahi from milk and preservation of the dahi.     

Inhibitor‐Assisted High‐Pressure Inactivation of Bacteria in Skim Milk

The combined inactivation effects of high hydrostatic pressure (HHP) and antimicrobial compounds (potassium sorbate and ε‐polylysine [ε‐PL]) on 4 different bacterial strains present in skim milk and the effect of these treatments on milk quality were investigated in this study. HHP treatment at 500 MPa for 5 min reduced the populations of Escherichia coli, Salmonella enterica Typhimurium, Listeria monocytogenes, and Staphylococcus aureus from 6.5 log colony‐forming units (CFUs) or higher to less than 1 log CFU/mL. Compared to HHP alone, HHP with potassium or ε‐PL resulted in significantly higher reductions in the bacterial counts. After 5 min of treatment with HHP (500 MPa) and ε‐PL (2 mg/mL), no growth of E. coli, S. enterica Typhimurium, or L. monocytogenes in skim milk was observed during 15 d of refrigerated storage (4 ± 1 °C). Scanning electron microscopy analysis revealed that the synergistic treatments caused more serious damage to the bacterial cell walls. Quality assessments of the treated samples indicated that the combined treatments did not influence the color, the turbidity, the concentrations of –SH group of the proteins, or the in vitro digestion patterns of the milk. This study demonstrates that HHP with potassium or ε‐PL may be useful in the processing of milk or milk‐containing foods.     

Inhibition of Listeria monocytogenes in cold smoked salmon by addition of sakacin P and/or liveLactobacillus sakei cultures

Listeria monocytogenes is the causative agent of food-related listeriosis. It has the ability to grow in vacuum-packaged food at chiller temperatures and is relatively tolerant to salt and low pH, thus it is difficult to control its growth in food.Listeria monocytogenes was added to vacuum-packed cold smoked salmon together with known concentrations of the bacteriocin sakacin P or nisin and/or one of two isogenic strains of the lactic acid bacteriaLactobacillus sakei . The vacuum-packaged salmon samples were incubated at 10°C for 4 weeks. Of the isogenic L. sakei strains, one produced sakacin P and the other did not. Both sakacin P and nisin had an initial inhibiting effect on growth of L. monocytogenes. Addition of the sakacin P-producing or the non-producing L. sakei had a bacteriostatic effect on L. monocytogenes during the complete storage period. When the sakacin P-producing L. sakei culture was added to vacuum-packed cold smoked salmon together with sakacin P, a bacteriocidal effect on L. monocytogenes was observed.     

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, C_8:0) 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 (10⁶ CFU/ml) containing 0, 25, or 50 mM of CA or MC. At 37°C, all the treatments, excepting 25 mm CA, reduced the population of both pathogens by approximately 5.0 log CFU/ml in 6 h. At 24 h of storage at 8°C, MC at both levels and CA at 50 mM decreased L. monocytogenes and E. coli O157:H7, respectively by >5.0 log CFU/ml. At 48 h of 4°C storage, populations of L. monocytogenes and E. coli O157:H7 were decreased to below detection level (enrichment negative) by 50 mm 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.     

Preliminary characterization of bacteriocins from Lactococcus lactis,Enterococcus faecium and Enterococcus mundtii strains isolated from turbot (Psetta maxima)

The aim of this work was the characterization of new strains of lactic acid bacteria (LAB) from farmed fish and with potential application as biopreservatives against both Listeria monocytogenes and Staphylococcus aureus. Twenty-five strains of LAB isolated from the muscle of farmed turbot were investigated. Genetic identification of the bacteriocin-producing LAB strains was performed by means of a PCR method using novel BAL1/BAL2 16S ribosomal-RNA-targeted primers. Maximum bacteriocin production by Lactococcus lactis ssp. lactis USC-39, Enterococcus faecium USC-46 and Enterococcus mundtii USC-51 was detected in the stationary phase of growth. Both acidification and the production of hydrogen peroxide by LAB were ruled out as the source of the inhibition. In contrast, the antimicrobial activity of all three LAB strains was inactivated by the addition of proteinase K, thus confirming the proteinaceous nature of the inhibition. The activity against L. monocytogenes was maintained in the 3.5–5.5 or 3.5–6.5 pH range, depending on the LAB strain. Likewise, inhibition of S. aureus strains was observed in the 3.5–4.5 and in the 3.5–5.5 pH ranges, depending on the LAB strain and on the S. aureus strain tested. Bacteriocin activity was stable in all three strains after heating the cell-free extract for 60 min at 100 °C, or even for 15 min at 121 °C, in all the three LAB strains. The acidic and heat-resistant bacteriocins produced by the three LAB strains isolated from turbot, able to inhibit the growth of both L. monocytogenes and S. aureus may find application as biopreservatives in fermented and/or heated food products.     

Determination and validation of D-values for Listeria monocytogenes and Shiga toxin–producing Escherichia coli in cheese milk

Certain cheeses can be legally produced in the United States using raw milk, but they must be aged for at least 60 d to reduce pathogen risks. However, some varieties, even when aged for 60 d, have been shown to support growth of Listeria monocytogenes or survival of Shiga toxin–producing Escherichia coli (STEC). Thermization, as a subpasteurization heat treatment, has been proposed as a control to reduce the risk of pathogens in raw cheese milk while retaining some quality attributes in the cheese. However, the temperature and time combinations needed to enhance safety have not been well characterized. The objective of this research was to determine and validate decimal reduction values (D-values) for L. monocytogenes and STEC at thermization temperatures 65.6, 62.8, and 60.0°C; a D-value at 57.2°C was also determined for L. monocytogenes only. Nonhomogenized, pasteurized whole-milk samples (1 mL) were inoculated with 8-log cfu/mL L. monocytogenes or STEC (5- or 7-strain mixtures, respectively), vacuum-sealed in moisture-impermeable pouches, and heated via water bath submersion. Duplicate samples were removed at appropriate intervals and immediately cooled in an ice bath. Surviving bacteria were enumerated on modified Oxford or sorbitol MacConkey overlaid with tryptic soy agar to aid in the recovery of heat-injured cells. Duplicate trials were conducted, and survival data were used to calculate thermal inactivation rates. D_65.6°C-, D_62.8°C-, and D_60.0°C-values of 17.1 and 7.2, 33.8 and 16.9, and 146.6 and 60.0 s were found for L. monocytogenes and STEC, respectively, and a D_57.2°C-value of 909.1 s was determined for L. monocytogenes. Triplicate validation trials were conducted for each test temperature using 100 mL of milk inoculated with 3 to 4 log cfu/mL of each pathogen cocktail, A 3-log reduction of each pathogen was achieved faster in larger volumes than what was predicted by D-values (D-values were fail-safe). Data were additionally compared with published results from 21 scientific studies investigating L. monocytogenes and STEC in whole milk heated to thermization temperatures (55.0–71.7°C). These data can be used to give producers of artisanal raw-milk cheese flexibility in designing thermal processes to reduce L. monocytogenes and STEC populations to levels that are not infectious to consumers.     

Response of Listeria monocytogenes and Vibrio parahaemolyticus to High Hydrostatic Pressure

Listeria monocytogenes Scott A and CA, were subjected at 23°C to hydrostatic pressures ranging from 2,380 to 3,400 atm and Vibrio parahaemolyticus T-3765-1 from 680 to 1,700 atm. For L. monocytogenes Scott A, pressurization in ultra-high temperature-processed (UHT) milk and raw milk appeared to provide a protective effect and lessened cell death as compared to pressurization in phosphate-buffered saline (100 mM, pH 7.0). A population of about 10⁶ CFU/mL L. monocytogenes was killed by exposure to 3,400 atm within 80 min at 23°C in UHT milk. A population of about 10⁶ CFU/mL V. parahaemolyticus was killed by exposure to 1,700 atm within 10 min at 23°C in clam juice.     

Efficacy of bacteriocin-containing cell-free culture supernatants from lactic acid bacteria to control Listeria monocytogenes in food

Consumer demands have led to an increased interest in the use of natural antimicrobials for food protection. With the objective of developing novel products for enhancing the microbial safety of food, we have tested cell-free culture supernatants (CFS's) of eight antagonistic bacterial strains for their efficacy to inhibit Listeria monocytogenes in different food matrices. The antagonistic strains represented different members of the order Lactobacillales as well as one isolate of Staphylococcus sciuri and all showed strong inhibition of L. monocytogenes on agar plates. Cell-free supernatants were obtained after growing the bacteria in a yeast extract-glucose broth. In six of the CFS's, different class IIa bacteriocins, namely leucocin A, leucocin B, mundticin L, pediocin PA-1, sakacin A, and sakacin X, were identified as the major anti-listerial compounds. For the other two strains, the active substances could not be ascertained conclusively. The minimal effective concentration (MEC) of the individual CFS's to achieve a 2.3 log₁₀ reduction of L. monocytogenes was determined in culture broth, whole milk, and ground beef at 4 °C. While all bacteriocin-containing CFS's were effective in broth at concentrations from 52 to 205 AU/ml, significant higher concentrations were needed when applied in food. Best results were obtained using CFS's containing pediocin PA-1, that displayed only three- and ten-times higher MEC's in milk (307 AU/ml) and ground meat (1024 AU/g) compared to broth, respectively. A twenty-fold increase in the MEC (2048 AU/ml) was observed for a mundticin L-containing fermentate, and a CFS containing leucocin A and B was inactivated more than fifty-fold (> 1280 AU/ml) in both food matrices. Remarkably, the sakacin A and sakacin X containing CFS's displayed very selective inactivation rates, in which sakacin A was only effective in meat (512 AU/g), while sakacin X was only effective in milk (2048 AU/ml). In all cases, inhibition of L. monocytogenes was only transient and surviving or resistant bacteria started growing after prolonged storage. These results highlight the importance of careful testing the effectiveness of bacteriocins in the food systems for which they are intended to be applied against the selected target and non-target bacteria. Furthermore, the outgrowth of surviving or resistant bacterial populations points out that the tested bacteriocins are not suited to assure full inhibition of L. monocytogenes in a food product, if not applied in combination with additional preservative measures.     

Growth and survival ofListeria monocytogenesin two traditional foods from the United Arab Emirates

The survival ofListeria monocytogenesin two foods of Arabic origin, namely labneh and houmos, was studied. Counts ofL. monocytogenesin excess of 10000 cfu g⁻¹from labneh (pH 3.8) were reduced to zero within 72 h at 4 and 10°C, but increased survival was noted at pH 4.5, especially in unsalted labneh; at 30°C, no organisms could be detected 24 h after inoculation. From a starting count of 27000 cfu g⁻¹,L. monocytogeneswas able to survive on houmos throughout the anticipated shelf life (3 days); when houmos was stored at 4 and 10°C under olive oil, the numbers ofListeriadeclined slowly over 3 days. It was concluded that care should be taken to avoid the contamination of houmos withListeriaduring preparation in the kitchen.