Combined action of nisin and carvacrol on Bacillus cereus and Listeria monocytogenes

Nisin, a small antimicrobial protein, was tested for its bactericidal action against Listeria monocytogenes and Bacillus cereus and a typical biphasic reduction of the viable count was observed. The reduction was most fast during the first 10 min of exposure, while the viable count remained stable in the last part of the exposure period. Bacillus cereus was more sensitive towards nisin than L. monocytogenes and the inhibitory effect of nisin was stronger towards cells cultivated and exposed at 8 degrees C than towards cells cultivated and exposed at 20 degrees C. Combining nisin with sublethal doses of carvacrol resulted in an increased reduction in the viable count of both organisms, indicating synergy between nisin and carvacrol. Addition of lysozyme as a third preservative factor increased the synergistic effect between nisin and carvone, especially in the last part of the exposure period.     

Combined effect of mild heat and acetic acid treatment for inactivating Escherichia coli O157:H7, Listeria monocytogenes and Salmonella typhimurium in an asparagus puree

AIMS: This study was conducted to validate combined heat and acid treatments for inactivating Escherichia coli O157:H7, Listeria monocytogenes and Salmonella typhimurium in an acidified brine containing, or pickled, asparagus model food. METHODS AND RESULTS: A mixture of three strains of E. coli O157:H7, L. monocytogenes and S. typhimurium were inoculated onto pickled asparagus samples. Combinations of various concentrations of acetic acid [0%, 0.25%, 0.5%, 0.75%, 1%, 1.5% and 2% (v/v)] and various temperatures (40 degrees C, 50 degrees C, 60 degrees C and 75 degrees C) were investigated. Following treatment, asparagus samples were stored at room temperature and enumerated at 0, 0.5, 1, 2 and 3 days. Heat and acetic acid treatments were synergistic. The inhibitory effects of these combined treatments on the tested foodborne pathogens were also effective during storage. Loss of green colour in the pickled asparagus significantly increased with increasing concentrations of acetic acid. CONCLUSIONS: Using a combination of mild heat and acetic acid treatments can successfully control E. coli O157:H7, L. monocytogenes and S. typhimurium in pickled asparagus, combinations of heat and acid are synergistic and effective treatments can be selected to reduce adverse effect on colour which occur during product storage. SIGNIFICANCE AND IMPACT OF THE STUDY: Mild heating plus acetic acid treatment are synergistic, so combined treatments can be developed, which would lower the temperature and amount of acetic acid required for minimally processed vegetables while maintaining pathogen control.     

Enhanced levels of cold shock proteins in Listeria monocytogenes LO28 upon exposure to low temperature and high hydrostatic pressure

Listeria monocytogenes is a psychrotrophic food-borne pathogen that is problematic for the food industry because of its ubiquitous distribution in nature and its ability to grow at low temperatures and in the presence of high salt concentrations. Here we demonstrate that the process of adaptation to low temperature after cold shock includes elevated levels of cold shock proteins (CSPs) and that the levels of CSPs are also elevated after treatment with high hydrostatic pressure (HHP). Two-dimensional gel electrophoresis combined with Western blotting performed with anti-CspB of Bacillus subtilis was used to identify four 7-kDa proteins, designated Csp1, Csp2, Csp3, and Csp4. In addition, Southern blotting revealed four chromosomal DNA fragments that reacted with a csp probe, which also indicated that a CSP family is present in L. monocytogenes LO28. After a cold shock in which the temperature was decreased from 37 degrees C to 10 degrees C the levels of Csp1 and Csp3 increased 10- and 3.5-fold, respectively, but the levels of Csp2 and Csp4 were not elevated. Pressurization of L. monocytogenes LO28 cells resulted in 3.5- and 2-fold increases in the levels of Csp1 and Csp2, respectively. Strikingly, the level of survival after pressurization of cold-shocked cells was 100-fold higher than that of cells growing exponentially at 37 degrees C. These findings imply that cold-shocked cells are protected from HHP treatment, which may affect the efficiency of combined preservation techniques.     

Combined action of S-carvone and mild heat treatment on Listeria monocytogenes Scott A

The combined action of the plant-derived volatile, S-carvone, and mild heat treatment on the food-borne pathogen, Listeria monocytogenes, was evaluated. The viability of exponential phase cultures grown at 8 degrees C could be reduced by 1.3 log units after exposure to S-carvone (5 mmol l-1) for 30 min at 45 degrees C, while individual treatment with S-carvone or exposure to 45 degrees C for 30 min did not result in a loss in viability. Other plant-derived volatiles, namely carvacrol, cinnamaldehyde, thymol and decanal, were also found to reduce the viability of L. monocytogenes in combination with the same mild heat treatment at concentrations of 1.75 mmol l-1, 2.5 mmol l-1, 1.5 mmol l-1 and 2 mmol l-1, respectively. These findings show that essential oil compounds can play an important role in minimally processed foods, and can be used in the concept of Hurdle Technology to reduce the intensity of heat treatment or other individual hurdles.     

Environmental factors influencing the inactivation of Listeria monocytogenes by pulsed electric fields

AIMS: To investigate the influence of the growth phase, growth temperature, storage time, pH and aw of the treatment medium on the resistance of Listeria monocytogenes to pulsed electric fields (PEF). METHODS AND RESULTS: Square wave pulses of 2 micros at a frequency of 1 Hz and 25 and 28 kV cm(-1) were used. Cells were more PEF resistant in the stationary than in the exponential phase at both incubation temperatures investigated (4 and 35 degrees C). Cells grown at 4 degrees C were more PEF sensitive than cells grown at 35 degrees C independent of the growth phase. After a treatment of 25 kV cm(-1) and 800 micros, 1.48, 3.86 and 5.09 log10 cycles of inactivation were obtained at pH 7.0, 5.4 and 3.8, respectively. A reduction in the aw of the treatment medium protected cells against PEF treatments. CONCLUSIONS: The PEF resistance of L. monocytogenes depended on different environmental factors. The influence of growth conditions and treatment medium characteristics should be known and controlled to obtain reproducible and reliable PEF inactivation data. SIGNIFICANCE AND IMPACT OF THE STUDY: Erroneous conclusions and misinterpretation of results are possible if factors affecting the PEF resistance of L. monocytogenes are not considered during PEF inactivation studies.     

Synergistic antibacterial action of heat in combination with nisin and magainin II amide

Lactobacillus plantarum has been exposed to mild heat at temperatures between 48 and 56 °C in combination with low concentrations of the lantobiotic nisin in different sequential set-ups. Exposure to heat and nisin caused synergistic reductions of Lact. plantarum viability. Efficient antimicrobial action was dependent on the growth state of the culture as well as on levels and sequences of treatment applications. Listeria monocytogenes and Escherichia coli were treated at 55 °C in the presence of magainin II amide. Synergistic reductions in viable counts could be observed for L. monocytogenes and, after prolonged exposure, also for E. coli . The bacterial membrane could be identified by fluorometry and flow cytometry as an important target of applied treatment combinations.     

The differential adherence capabilities of two Listeria monocytogenes strains in monoculture and multispecies biofilms as a function of temperature

AIMS: To determine the differential adherence capabilities at three different temperatures of Listeria monocytogenes Scott A, a clinical food pathogen, and L. monocytogenes FM876, a persistent strain from a milk-processing environment, to stainless steel. METHODS AND RESULTS: Differential adherence was investigated by submerging stainless steel coupons in both 48-h Listeria monocultures and mixed cultures additionally containing Staphylococcus xylosus DP5H and Pseudomonas fragi ATCC 4973. Immunofluorescent microscopy and image analysis techniques were utilized to identify and quantify the L. monocytogenes cells adhering to the steel at 4 degrees C, 18 degrees C and 30 degrees C. The monoculture biofilms consistently contained greater L. monocytogenes numbers than the multispecies biofilms, with the persistent strain FM876 showing significantly greater adherence than strain Scott A. Optimum adherence occurred at 18 degrees C in monoculture biofilms. CONCLUSION: L. monocytogenes strains exhibit differential, temperature-dependent, adherence to stainless steel. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate temperature dependent biofilm adherence and support previous findings that persistent strains exhibit increased adherence capability.     

Effects of acidified sodium chlorite, cetylpyridinium chloride and hot water on populations of Listeria monocytogenes and Staphylococcus aureus on beef

AIMS: The present study was designed to determine the individual and combined effects of acidified sodium chlorite (ASC, 0.1%, 24 +/- 1 degrees C), cetylpyridinium chloride (CPC, 0.5%, 24 +/- 1 degrees C) and hot water (HW, 93 +/- 1 degrees C) treatments on the survival of Listeria monocytogenes and Staphylococcus aureus. METHODS AND RESULTS: Beef samples inoculated with L. monocytogenes and S. aureus were treated with nine different applications singly or in combination. Treatment groups comprised (i) untreated control; (ii) sterile tap water; (iii) 0.1% ASC; (iv) 0.5% CPC; (v) HW; (vi) HW followed by 0.1% ASC; (vii) HW followed by 0.5% CPC; (viii) 0.1% ASC followed by HW; (ix) 0.5% CPC followed by HW. Compared with the untreated control group, the reductions in L. monocytogenes populations were 1.14-2.31 log CFU g(-1), while the reductions in S. aureus populations were 0.83-2.74 log CFU g(-1) on day 0. CONCLUSION: The reduction effect that occurred after combined treatment with ASC followed by HW, HW followed by ASC, CPC followed by HW and HW followed by CPC was found to be significantly greater (P < 0.05) than after treatment with ASC and CPC alone on days 0, 2 and 4 of storage. SIGNIFICANCE AND IMPACT OF THE STUDY: ASC, CPC and HW treatments can be used to reduce L. monocytogenes and S. aureus, which would provide an additional measure of safety on the production line.     

Detection and characterization of the novel bacteriocin entomocin 9, and safety evaluation of its producer, Bacillus thuringiensis ssp. entomocidus HD9

AIMS: To identify and characterize new bacteriocins from a collection of 41 strains belonging to 27 subspecies of Bacillus thuringiensis, and to evaluate the safety of the producers. METHODS AND RESULTS: Bacillus thuringiensis ssp. entomocidus HD9 produced in the culture supernatant an antimicrobial activity against Gram-positive bacteria including Listeria monocytogenes, one of four pathogenic Pseudomonas aeruginosa and several fungi. Production of the antibacterial activity, named entomocin 9, started during mid-logarithmic growth reaching its maximum at the early stationary phase. Entomocin 9 retained more than 72% of activity after incubation for 20 min at 121 degrees C. Activity was lost after proteinase K treatment, it was stable in a pH range between 3 and 9, and resistant to lyophilization. After partial purification with ammonium sulphate precipitation followed by gel-filtration and anion-exchange chromatography, an active protein of ca 12.4 kDa was isolated. The mode of action of entomocin 9 was bactericidal and caused cell lysis of growing cells. Despite the presence of a range of virulence related genes, including haemolysin BL, nonhaemolytic enterotoxin, cytotoxin K and several hydrolytic activities, B. thuringiensis HD9 was not toxic against Vero cells. CONCLUSIONS: Entomocin 9 is a novel heat-stable, bacteriocin produced by B. thuringiensis HD9. The absence of toxicity against Vero cells suggests the suitability of strain HD9 for a safe application in antimicrobial treatments. SIGNIFICANCE AND IMPACT OF THE STUDY: New finding on entomocin 9 would make B. thuringiensis attractive in biotechnological applications as an antimicrobial agent in agriculture and food industry.     

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.     

Sensitivity of nisin-resistant Listeria monocytogenes to heat and the synergistic action of heat and nisin

Nisin, a bacteriocin produced by some strains of Lactococcus lactis, acts against foodborne pathogen Listeria monocytogenes. A single exposure of cells to nisin can generate nisin-resistant (Nisr) mutants, which may compromise the use of nisin in the food industry. The objective of this research was to compare the heat resistance of Nisr and wild type (WT) Listeria monocytogenes. The synergistic effect of heat-treatment (55 degrees C) and nisin (500 IU ml-1) on the Nisr cells and the WT L. monocytogenes Scott A was also studied. When the cells were grown in the absence of nisin, there was no significant (alpha = 0.05) difference in heat resistance between WT and Nisr cells of L. monocytogenes at 55, 60 and 65 degrees C. However, when the Nisr cells were grown in the presence of nisin, they were more sensitive to heat at 55 degrees C than the WT cells. The D-values at 55 degrees C were 2.88 and 2.77 min for Nisr ATCC 700301 and ATCC 700302, respectively, which was significantly (alpha = 0.05) lower than the D-value for WT, 3.72 min. When Nisr cells were subjected to a combined treatment of heat and nisin, there was approximately a four log reduction during the first 7 min of treatment.     

Enhanced inactivation of Listeria monocytogenes by nisin in the presence of ethanol

AIMS: The effect of combinations of nisin and ethanol on the survival of Listeria monocytogenes was investigated. METHODS AND RESULTS: Killing by nisin was enhanced during simultaneous exposure to ethanol (2-7% v/v). For example, while 10 IU ml(-1) nisin reduced viability by 1 log unit in 20 min, a combination of this antimicrobial peptide and 5% ethanol, reduced numbers of surviving cells by 3 log units. Increasing the concentrations of either ethanol (2-7%) or nisin (10-50 IU ml(-1)) led to increased cell death with synergy being demonstrated for all combinations tested and at a range of temperatures from 5 to 37 degrees C. CONCLUSIONS: Ethanol can act synergistically with nisin to reduce the survival of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Combinations of ethanol and nisin may be feasible as an effective way of controlling this pathogen in the food processing environment.     

Adaptation of the food-borne pathogen Bacillus cereus to carvacrol

Carvacrol, a natural antimicrobial compound present in the essential oil fraction of oregano and thyme, is bactericidal towards Bacillus cereus. A decrease of the sensitivity of B. cereus towards carvacrol was observed after growth in the presence of non-lethal carvacrol concentrations. A decrease of the melting temperature (Tm) of membranes from 20.5 degrees C to 12.6 degrees C was the immediate effect of the addition of carvacrol. Cells adapted to 0.4 mM carvacrol showed a lower membrane fluidity than nonadapted cells. Adaptation of 0.4 mM carvacrol increased the Tm from 20.5 degrees C to 28.3 degrees C. The addition of carvacrol to cell suspensions of adapted B. cereus cells decreased Tm again to 19.5 degrees C, approximately the same value as for the non-adapted cells in the absence of carvacrol. During adaptation, changes in the fatty acid composition were observed. The relative amount of iso-C13:0, C14:0, and iso-C15:0 increased and cis-C16:1 and C18:0 decreased. The head-group composition also changed, two additional phospholipids were formed and one phospholipid was lacking in the adapted cells. It could be concluded that B. cereus adapts to carvacrol when present at non-lethal concentrations in the growth medium by lowering its membrane fluidity by changing the fatty acid and headgroup composition.     

Antimicrobial effects of mustard flour and acetic acid against Escherichia coli O157:H7, Listeria monocytogenes,and Salmonella enterica serovar Typhimurium

This study was designed to investigate the individual and combined effects of mustard flour and acetic acid in the inactivation of food-borne pathogenic bacteria stored at 5 and 22 degrees C. Samples were prepared to achieve various concentrations by the addition of acetic acid (0, 0.5, or 1%) along with mustard flour (0, 10, or 20%) and 2% sodium chloride (fixed amount). Acid-adapted three-strain mixtures of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium strains (10(6) to 10(7) CFU/ml) were inoculated separately into prepared mustard samples stored at 5 and 22 degrees C, and samples were assayed periodically. The order of bacterial resistance, assessed by the time required for the nominated populations to be reduced to undetectable levels against prepared mustards at 5 degrees C, was S. enterica serovar Typhimurium (1 day) < E. coli O157:H7 (3 days) < L. monocytogenes (9 days). The food-borne pathogens tested were reduced much more rapidly at 22 degrees C than at 5 degrees C. There was no synergistic effect with regard to the killing of the pathogens tested with the addition of 0.5% acetic acid to the mustard flour (10 or 20%). Mustard in combination with 0.5% acetic acid had less bactericidal activity against the pathogens tested than did mustard alone. The reduction of E. coli O157:H7 and L. monocytogenes among the combined treatments on the same storage day was generally differentiated as follows: control < mustard in combination with 0.5% acetic acid < mustard alone < mustard in combination with 1% acetic acid < acetic acid alone. Our study indicates that acidic products may limit microbial growth or survival and that the addition of small amounts of acetic acid (0.5%) to mustard can retard the reduction of E. coli O157:H7 and L. monocytogenes. These antagonistic effects may be changed if mustard is used alone or in combination with >1% acetic acid.     

Carvacrol and cinnamic acid inhibit microbial growth in fresh-cut melon and kiwifruit at 4 degrees and 8 degrees C

AIM: To establish whether or not carvacrol and cinnamic acid delay microbial spoilage of fresh-cut fruit. METHODS AND RESULTS: Dipping of fresh-cut kiwifruit in carvacrol solutions at 5-15 mM reduced total viable counts from 6.6 to < 2 log cfu g-1 for 21 d at 4 degrees C; however, undesirable colour and odour changes were also observed. Treatment with 1 mM of carvacrol or cinnamic acid reduced viable counts on kiwifruit by 4 and 1.5 log cfu g-1 for 5 d at 4 degrees C and 8 degrees C, respectively. Treatment of fresh-cut honeydew melon with 1 mM of carvacrol or cinnamic acid extended the lag phase of the microbial flora from less than 1 d in the untreated controls to 3 d at 8 degrees C and 5 d at 4 degrees C. Viable counts on the treated melon were 6 log cfu g-1 lower on Day 3 at 8 degrees C and 4 log cfu g-1 lower on Day 5 at 4 degrees C, compared with the untreated controls. IMPACT OF THE STUDY: Treatment with 1 mM of carvacrol or cinnamic acid delays spoilage of fresh-cut kiwifruit and honeydew melon at chill temperatures without adverse sensory consequences.     

Relationship between inactivation kinetics of a Listeria monocytogenes suspension by chlorine and its chlorine demand

AIMS: Chlorine demand by Listeria monocytogenes cells and inactivation of L. monocytogenes by chlorine (0.6-1.0 mg l(-1)) at different temperatures (4, 20 and 30 degrees C) have been investigated in a batch reactor. METHODS AND RESULTS: Chlorine demand depended on the microbial concentration and was independent on the initial chlorine concentration and temperature. Chlorine decay was modelled by the addition of two first-order decay equations. Inactivation of L. monocytogenes by chlorine depended on the initial microbial concentration, initial chlorine concentration and temperature. A mathematical model based on a biphasic inactivation properly described survival curves of L. monocytogenes and a tertiary model was developed that satisfactorily predicted the inactivation of L. monocytogenes by different concentrations of initial chlorine at different temperatures. CONCLUSIONS: Both available chlorine decay and inactivation of L. monocytogenes by chlorine were biphasic and can be modelled by a two-term exponential model. SIGNIFICANCE AND IMPACT OF THE STUDY: The biphasic nature of survival curves of L. monocytogenes did not reflect the effect of a change of available chlorine concentration during the treatment. The microbial inactivation was caused by successive reactions that occur after the consumption of the chlorine by the bacterial cell components.     

Characterization of an antibacterial peptide produced by Brevibacterium linens

AIMS: The aim of this research was to investigate the antimicrobial activity produced by Brevibacterium linens ATCC 9175. METHODS AND RESULTS: A bacteriocin produced by the red smear cheese bacterium B. linens ATCC 9175 was identified. The antimicrobial activity was first produced at the exponential growth phase. A crude bacteriocin obtained from the culture supernatant fluid was inhibitory to some indicator strains. It inhibited the growth of Listeria monocytogenes ATCC 7644, B. linens ATCC 9172 and Corynebacterium fimi NCTC 7547, but was inactive against the Gram-negative bacteria and yeast tested. The bacteriocin was stable at 30 degrees C but the activity was lost when the temperature reached 50 degrees C. It was sensitive to the proteolytic action of trypsin, papain and pronase E and was active between pH 6.0 and 9.0. The bacteriocin was bactericidal to L. monocytogenes at 40 AU ml(-1). Bacteriostasis was observed for a low dose of bacteriocin (20 AU ml(-1)). CONCLUSIONS: An antibacterial peptide produced by B. linens was characterized, presenting potential for use as a biopreservative in food systems. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of a novel bacteriocin active against L. monocytogenes addresses an important aspect of food protection against pathogens and spoilage micro-organisms.     

Behaviour of Listeria monocytogenes under combined chilling processes

The behaviour of Listeria monocytogenes under chilling processes was investigated. Growth kinetics were measured at 7 degrees C in TSBYE culture medium as a function of pH (7.2 and 6.2), pre-incubation temperatures (4 or 7 degrees C), cooling (0.05 or 0.1 degree C min-1) and freezing (0 and -5 degrees C) treatments. Growth curves generated were fitted by Gompertz and Baranyi functions. The Baranyi function gave better parameter estimation values than the Gompertz equation which over-estimated the specific growth rate values. Listeria monocytogenes grew at 7 degrees C without a lag phase, except when the sub-culture was performed at 37 degrees C, whereas the specific growth rate was affected by the chilling processes. In fact, L. monocytogenes grew slightly faster at 7 degrees C when a 4 degrees C pre-incubation treatment was applied than with a 7 degrees C pre-incubation treatment. These results suggest that to mimic the processes of contamination in industry, predictive microbiology studies with L. monocytogenes should be performed with organisms cultured at low temperatures.     

Influence of interactions between temperature,ferric ammonium citrate and glycine betaine on the growth of Listeria monocytogenes in a defined medium

AIMS: To investigate interactions, if any, between temperature, ferric ammonium citrate and glycine betaine on the growth of Listeria monocytogenes in modified Pine's medium (Pine et al. 1989). METHODS AND RESULTS: Modified Pine's medium containing 0, 0.044, 0.088 or 0.176 g l(-1) ferric ammonium citrate, and 0 or 1 mM glycine betaine, was inoculated with each of two L. monocytogenes strains and incubated at 4, 25 or 37 degrees C. The optical density at 600 nm, and cell numbers, were determined at appropriate time intervals. At 4 degrees C, but not other temperatures, increasing ferric ammonium citrate resulted in improved growth in the absence, but not the presence, of glycine betaine. The presence of glycine betaine was inhibitory at 25 and 37 degrees C, but not at 4 degrees C. CONCLUSIONS: Interactions affecting the growth kinetics of L. monocytogenes were apparent between the parameters investigated. SIGNIFICANCE AND IMPACT OF THE STUDY: Limitations on the use of modified Pine's medium, and the significance of iron metabolism at lower temperatures, were revealed.     

Recovery of the viability of Chinese hamster V79-4 cells after combined exposure to hyperthermia and radiation

The survival of cells overheated (42 degrees C) before gamma irradiation is increased by holding them in the growth medium at 37 degrees C before treatment with hypertonic NaCl solution. The substantial synergistic effect of hyperthermia and radiation takes place when the cells are treated with a 1.5 M NaCl solution immediately after the combined action of these inactivating factors. The synergistic effect is decreased by holding the cells in the nutrient medium at 37 degrees C for 4 hours before hypertonic treatment.