Evaluation of PTR-ToF-MS as a tool to track the behavior of hop-derived compounds during the fermentation of beer.

Hop-derived volatile organic compounds (VOCs) play an important role in the flavor and aroma of beer, despite making up a small percentage of the overall profile. To understand the changes happening during fermentation, proton transfer reaction-time of flight-mass spectrometry (PTR-ToF-MS) was applied for the first time in brewing science to directly measure the changes in hop-derived VOCs during the fermentation of four different worts containing one of two aroma hops in combination with one of two yeast biotypes. PTR-ToF-MS successfully detected and tracked mass-to-charge ratios (m/z) arising from interactions between the different yeast strains and the hop cultivars. Differences were observed in the dynamic VOC profiles between different beer treatments for m/z such as m/z 145.121 (ethyl hexanoate) and m/z 173.153 (isoamyl isovalerate or ethyl octanoate). The ability to monitor changes in VOCs during fermentation provides valuable information on the priority of production and transformation reactions by yeast.

The effect of alginate lyase on the gentamicin resistance of Pseudomonas aeruginosa in mucoid biofilms.

AIMS: Pseudomonas aeruginosa can secrete large amounts of alginate during chronic infections and this has been associated with high resistance to antibiotics. The major aim of this study was to investigate whether degradation of extracellular alginate by alginate lyase would increase the sensitivity of Ps. aeruginosa to gentamicin, an aminoglycoside antibiotic. METHODS AND RESULTS: Degradation of alginate from Ps. aeruginosa was monitored using a spectrometric assay. Alginate lyase depolymerized alginate, but calcium and zinc cations at concentrations found in the cystic fibrosis lung reduced enzyme activity. Biofilms formed on agar were partially degraded by alginate lyase, but staining with crystal violet showed that the biomass of biofilms grown in liquid was not significantly affected by the enzyme. Viability testing showed that the sensitivity to gentamicin of biofilm bacteria and of bacteria released from biofilms was unaffected by alginate lyase. CONCLUSIONS: Our results show that at least under the conditions used here alginate lyase does not affect gentamicin resistance of Ps. aeruginosa. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study indicates that alginate does not contribute to resistance to gentamicin and so does not provide support for the concept of treating patients with alginate lyase in order to increase the antibiotic sensitivity of Ps. aeruginosa.

Prediction of the amount and rate of histamine degradation by diamine oxidase (DAO).

Histamine is a biogenic amine that forms in a variety of foods and can cause food poisoning at high concentrations (>500 ppm). In situations where the formation of histamine in food cannot be prevented through refrigeration, diamine oxidase (DAO) enzyme may be used to degrade histamine to safe levels. The aims of this work were to apply DAO in model (buffer) and real (cooked tuna soup used in the manufacture of a fish paste product, Rihaakuru) systems, in order to obtain predictions for the rates and amounts of histamine degradation. The two systems were set up with a constant concentration of histamine (500 mg/L) and the DAO enzyme (2534 units/L) at a temperature of 37°C, agitation at 100 rpm and an incubation time of 10h with variable pH (5-7) and salt concentrations (1-5%). A total of 15 experiments were designed for each system using central composite design (CCD). The data from these experiments were fitted into regression models; initially the data were used to generate an exponential decline model and then the data from this were fitted into a secondary response surface model (RSM) to predict the rate and amount of histamine degradation by DAO. The model system results indicated that DAO activity was not significantly affected by salt (p>0.05), and that activity reached a maximum within the pH range of 6-6.5 with an optimum at pH 6.3. However, the results obtained with the tuna soup model showed that the optimum oxidation of histamine using DAO occurred between pH 6-7 and salt 1-3%. This study defined the conditions for the use of DAO to degrade 500 mg/L of histamine in tuna soup used to manufacture Rihaakuru. The models generated could also be used to predict the rate and amount of histamine degradation in other foods that have similar characteristics to tuna soup.

Effect of NaOH (caustic wash) on the viability, surface characteristics and adhesion of spores of a Geobacillus sp. isolated from a milk powder production line.

AIM: To investigate the viability, surface characteristics and ability of spores of a Geobacillus sp. isolated from a milk powder production line to adhere to stainless steel surfaces before and after a caustic (NaOH) wash used in clean-in-place regimes. METHODS AND RESULTS: Exposing sessile spores to 1% NaOH at 65°C for 30min decreased spore viability by two orders of magnitude. The zeta potential of the caustic treated spores decreased from -20 to -32 mV and they became more hydrophobic. Transmission electron microscopy revealed that caustic treated spores contained breaks in their spore coat. Under flow conditions, caustic treated spores suspended in 0·1 mol l(-1) KCl were shown to attach to stainless steel in significantly greater numbers (4·6 log(10) CFU cm(-2) ) than untreated spores (3·6 log(10) CFU cm(-2) ). CONCLUSIONS: This research suggests that spores surviving a caustic wash will have a greater propensity to attach to stainless steel surfaces. SIGNIFICANCE OF STUDY: The practice of recycling caustic wash solutions may increase the risk of contaminating dairy processing surfaces with spores.

Characterization of spore surfaces from a Geobacillus sp. isolate by pH dependence of surface charge and infrared spectra.

AIMS: The surfaces of spores from a Geobacillus sp. isolated from a milk powder production line were examined to obtain fundamental information relevant to bacterial spore adhesion to materials. MATERIALS AND RESULTS: The surfaces of spores were characterized using transmission electron microscopy and infrared spectroscopy. Thin sections of spores stained with ruthenium red revealed an exosporium with a hair-like nap around the spores. Attenuated total reflection infrared spectra of the spores exposed to different pH solutions on a ZnSe prism revealed that pH-sensitive carboxyl and phosphodiester groups associated with proteins and polysaccharides contributed to the spore's negative charge which was revealed by our previous zeta potential measurements on the spores. Lowering the pH to the isoelectric point of spores resulted in an increase in intensity of all spectral bands, indicating that the spores moved closer to the zinc selenide (ZnSe) surface as the charged surface groups were neutralized and the spore surface polymers compressed. The attachment of spores to stainless steel was threefold higher at pH 3 compared with pH 7. CONCLUSIONS: This research showed that spore attachment to surfaces is influenced by electrostatic interactions, surface polymer conformation and associated steric interactions. SIGNIFICANCE AND IMPACT OF THE STUDY: The adhesion of thermophilic spores is largely controlled by functional groups of surface polymers and polymer conformation.

Mechanisms of Cation Exchange by Pseudomonas aeruginosa PAO1 and PAO1 wbpL, a Strain with a Truncated Lipopolysaccharide.

The ability of bacterial cells to sequester cations is well recognized, despite the fact that the specific binding sites and mechanistic details of the process are not well understood. To address these questions, the cation-exchange behavior of Pseudomonas aeruginosa PAO1 cells with a truncated lipopolysaccharide (LPS) (PAO1 wbpL) and cells further modified by growth in a magnesium-deficient medium (PAO1 wbpL - Mg(2+)) were compared with that of wild-type P. aeruginosa PAO1 cells. P. aeruginosa PAO1 cells had a negative surface charge (zeta potential) between pH 11 and 2.2, due to carboxylate groups present in the B-band LPS. The net charge on PAO1 wbpL cells was increasingly positive below pH 3.5, due to the influence of NH(3)(+) groups in the core LPS. The zeta potentials of these cells were also measured in Na(+), Ca(2+), and La(3+) electrolytes. Cells in the La(3+) electrolyte had a positive zeta potential at all pH values tested. Growing P. aeruginosa PAO1 wbpL in magnesium-deficient medium (PAO1 wbpL - Mg(2+)) resulted in an increase in its zeta potential in the pH range from 3.0 to 6.5. In cation-exchange experiments carried out at neutral pH with either P. aeruginosa PAO1 or PAO1 wbpL, the concentration of bound Ca(2+) was found to decrease as the pH was reduced from 7.0 to 3.5. At pH 3.5, the bound Mg(2+) concentration decreased sharply, revealing the activity of surface sites for cation exchange and their pH dependence. Infrared spectroscopy of attached biofilms suggested that carboxylate and phosphomonoester functional groups within the core LPS are involved in cation exchange.

Recovery of spores from thermophilic dairy bacilli and effects of their surface characteristics on attachment to different surfaces.

Spores from four Geobacillus spp. were isolated from a milk powder manufacturing line in New Zealand. Liquid sporulation media produced spore yields of approximately 10(7) spores ml(-1); spores were purified using a two-phase system created with polyethylene glycol 4000 and 3 M phosphate buffer. The zeta potentials of the spores from the four isolates ranged from -10 to -20 mV at neutral pH, with an isoelectric point between pH 3 and 4. Through contact angle measurements, spores were found to be hydrophilic and had relative hydrophobicity values of 10 to 40%, as measured by the microbial adhesion to hexadecane assay. The most hydrophilic spore isolate with the smallest negative charge attached in the highest numbers to Thermanox and stainless steel (1 x 10(4) spores cm(-2)), with fewer spores attaching to glass (3 x 10(3) spores cm(-2)). However, spores produced by the other three strains attached in similar numbers (P > 0.05) to all substrata (approximately 1 x 10(3) spores cm(-2)), indicating that there was no simple relationship between individual physicochemical interactions and spore adherence. Therefore, surface modifications which limit the attachment of one strain may not be effective for all stains, and control regimens need to be devised with reference to the characteristics of the particular strains of concern.

The association of E. coli and soil particles in overland flow.

The removal of E. coli from overland flow under saturation-excess runoff conditions was investigated in experimental field plots that were 1 m wide and 5 m long. Variation in the attenuation of bacteria and distance transported was quantified under contrasting flow conditions. In addition, the impact of soil tillage upon microbial attenuation was examined by comparing results derived from grassed plots (intact) with those subject to tillage with the soil left bare (cultivated). For intact plots subjected to a flow of 2 L/min, 27% of the E. coli in the flow was removed after 5 m with removal following a logarithmic function with respect to distance. For the higher flow rates of 6 L/min and 20 L/min, no attenuation trend was observed over this distance. E. coli removal during flow across the cultivated plots was significantly greater compared to the intact plots. This was attributed to a greater infiltration rate in the cultivated plots (due to the tillage) which promoted a greater volume of flow to pass through the soil matrix, providing the opportunity for filtration and adsorption of microbes. Logarithmic trends with respect to distance were observed for all flow rates tested on the cultivated plots (2, 6 and 20 L/min). Total removal after 5 m at a flow rate of 2 L/min was 41% and again removal efficiency decreased as the flow rate increased. Analysis of the transported state of the E. coli revealed that the bacteria were being transported predominantly in particles less than 20 microm in diameter and were not attached to large (dense) soil particles. The limited removal (< 50%) of bacteria from overland flow under saturation-excess runoff conditions in these experiments appeared, therefore, to be primarily due to a lack of settling or deposition. Instead, most bacteria remained entrained within the overland flow down the length of the plots.

Numbers and transported state of Escherichia coli in runoff direct from fresh cowpats under simulated rainfall.

AIMS: To investigate the number of Escherichia coli in runoff derived directly from fresh cowpats and to determine if the E. coli are attached to dense particles, in flocs or as individual cells. METHODS AND RESULTS: Three cowpats were collected monthly from the same farm for 13 months and the number of E. coli in them estimated. A rainfall simulator was used to generate runoff from the individual cowpats, which was fractioned to determine the transported state of any E. coli present. The number of E. coli in the cowpat runoff was highly variable and was strongly correlated with the number of E. coli in the cowpat. Only a small percentage (approx. 8%) of the E. coli in runoff were attached to dense (>1.3 g ml(-1)) particles and there was no evidence of flocculation of the cells. CONCLUSIONS: Escherichia coli in runoff from cowpats are transported predominantly as individual cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Mitigation strategies to reduce the number of faecal bacteria in overland flow from agricultural land need to be designed to trap single bacterial cells.

Survival of Campylobacter jejuni in water: effect of grazing by the freshwater crustacean Daphnia carinata (Cladocera).

Environmental studies of the human-pathogenic bacterium Campylobacter jejuni have focused on linking distributions with potential sources. However, in aquatic ecosystems, the abundance of C. jejuni may also be regulated by predation. We examine the potential for grazing by the freshwater planktonic crustacean Daphnia carinata to reduce the survival of C. jejuni. We use a system for measuring grazing and clearance rates of D. carinata on bacteria and demonstrate that D. carinata can graze C. jejuni cells at a rate of 7% individual(-1) h(-1) under simulated natural conditions in the presence of an algal food source. We show that passage of C. jejuni through the Daphnia gut and incorporation into fecal material effectively reduces survival of C. jejuni. This is the first evidence to suggest that grazing by planktonic organisms can reduce the abundance of C. jejuni in natural waters. Biomanipulation of planktonic food webs to enhance Daphnia densities offers potential for reducing microbial pathogen densities in drinking water reservoirs and recreational water bodies, thereby reducing the risk of contracting water-borne disease.

Evaluation of the effectiveness of a commercially available defined substrate medium and enumeration system for measuring Escherichia coli numbers in faeces and soil samples.

AIMS: To determine if a commercially available defined substrate medium and enumeration system could be utilized as an effective and accurate means of enumerating Escherichia coli in environmental samples containing faeces and soil. METHODS AND RESULTS: The samples tested were either inoculated with laboratory grown E. coli or natural E. coli populations in cow faeces. The number of E. coli recovered from faeces and soil samples using the defined substrate medium and enumeration system and a miniaturized MPN method (using traditional media) was compared by analysing the difference between the two methods in relation to the mean. For four of five groups of samples analysed there was no significant difference in the number of E. coli recovered by the two methods (P > 0.05). In one batch the difference was 0.30 log, which while being statistically significant (P < 0.01) was not considered to be biologically significant. CONCLUSION: The commercially available enumeration system was significantly more precise than the miniaturized MPN method (P < 0.001). SIGNIFICANCE AND IMPACT OF THE STUDY: We conclude that the commercially available defined substrate medium and enumeration system is a suitable method for the measurement of E. coli numbers in faeces and soil samples and should provide advantages of increased precision and a reduction in laboratory analysis time.

Development of a method to quantify in vitro the synergistic activity of "natural" antimicrobials.

Despite numerous papers being published on the use of hurdle technology to control food-borne pathogens or spoilage organisms, there is no commonly accepted methodology to quantify the level of synergistic activity. This paper describes a method to quantify in vitro the synergistic activity of antibacterial agents against bacteria. Initially, a microtiter plate growth assay was used to determine the inhibitory concentrations of four "natural" antimicrobials (nisin, lauricidin, totarol, and the lactoperoxidase system (LPS)) against a panel of eight bacteria. Using the same microtiter system, the impact of various combinations of antimicrobials was assessed. The degree of synergy was based on the analysis of three criteria: (1) increase in lag phase, (2) reduction in culture density after 24 h, (3) and residual viability at 24 h. Only the lactoperoxidase system was active against all the Gram-positive and Gram-negative bacteria tested. Nisin, lauricidin, and totarol were only effective against the Gram-positive bacteria. The method successfully identified three combinations (nisin-lauricidin, LPS-nisin, and LPS-lauricidin) previously reported to have synergistic activity and highlighted the synergistic activity of two novel combinations (nisin-totarol and LPS-totarol). The development of a quick and reliable method to identify and quantify synergistic activity is a useful screening tool to establish preservative techniques that could have potential antimicrobial synergy in food-based systems.

Inactivation of Listeria monocytogenes/Flavobacterium spp. biofilms using chlorine: impact of substrate, pH, time and concentration.

AIMS: To determine the effect of chlorine on mixed bacterial biofilms on stainless steel (SS) and conveyor belt surfaces. METHODS AND RESULTS: Biofilms were exposed to pH-adjusted (6.5) and non-pH-adjusted solutions of chlorine (200, 400 and 600 ppm) for either 2, 10 or 20 min and survivors enumerated. There were significant differences in cell death relating to chlorine concentration and exposure time for the cells attached to the SS, with solutions adjusted to pH 6.5 being more effective at reducing numbers. In contrast, on conveyor belt surfaces cell numbers decreased by less than two logs after 20 min regardless of treatment. CONCLUSIONS: Chlorine effectiveness is dependent on its concentration, solution pH, exposure time, the nature of the surface and the microbial species present. SIGNIFICANCE AND IMPACT OF THE STUDY the interests of food safety it is important that sanitizer users are aware of the conditions that effect their performance.

Development of a technique to quantify the effectiveness of enrichment regimes in recovering "stressed" Listeria cells.

A rapid, reliable microwell plate method based on the most probable number (MPN) technique was used to determine the effectiveness of five enrichment regimes in the recovery and enumeration of Listeria spp. cells from five seafood products. The products tested were chosen to reflect conditions under which cells were exposed to the "stresses" associated with a variety of food-processing techniques, such as treatments involving an ethanol-based marinade, lowered pH (acetic acid), heat, sugar and salt brine (Gravilax), or frozen storage. Either Listeria monocytogenes and Listeria innocua were present in food samples as natural contaminants or L monocytogenes was added in the laboratory. Listeria repair broth (LRB), buffered Listeria enrichment broth, Listeria enrichment broth (LEB), Fraser broth, and University of Vermont modified Listeria enrichment broth were used to recover Listeria cells. The effectiveness of these enrichment regimes was found to be dependent on the type of stresses the cells had been exposed to. After exposure to ethanol, recovery of L monocytogenes cells was inhibited in enrichment regimes involving a nonselective period of resuscitation. On exposure to acetic acid, there were no significant differences (P < 0.05) between any of the regimes used. With heat-stressed cells, LRB recovered significantly fewer (P < 0.05) cells than did any other medium. On exposure to osmotic stress (elevated sugar and salt concentrations), LEB recovered the fewest cells. The largest number of cells was recovered from frozen fish (Hoki [Macruronus novazelandiae]) fillets with LRB. No single enrichment regime was consistently the most effective.

Survival of Listeria monocytogenes attached to stainless steel surfaces in the presence or absence of Flavobacterium spp.

Contaminated surfaces of food processing equipment are believed to be a significant source of Listeria monocytogenes to foods. However, very little is known about the survival of Listeria in processing environments. In a mixed bacterial biofilm of L. monocytogenes and Flavobacterium spp., the number of L. monocytogenes cells attaching to stainless steel increased significantly compared to when L. monocytogenes was in a pure culture. The L. monocytogenes cells in the mixed biofilms were also recoverable for significantly longer exposure periods. On colonized coupons held at 15 degrees C and 75% humidity, decimal reduction times were 1.2 and 18.7 days for L. monocytogenes in pure and mixed biofilms, respectively. With increasing exposure time, the proportion of cells that were sublethally injured (defined as an inability to grow on selective agar) increased from 8.1% of the recoverable cell population at day 0 to 91.4% after 40 days' exposure. At 4 and -20 degrees C, decimal reduction times for L. monocytogenes in pure culture were 2.8 and 1.4 days, respectively, and in mixed culture, 10.5 and 14.4 days, respectively. The enhanced colonization and survival of L. monocytogenes on "unclean" surfaces increase the persistence of this pathogen in food processing environments, while the increase in the percentage of sublethally injured cells in the population with time may decrease the ability of enrichment regimes to detect it.

The effect of 100% CO2 on the growth of nonproteolytic Clostridium botulinum at chill temperatures.

The growth of a cocktail of spores from six nonproteolytic Clostridium botulinum type B and E isolates at 5 and 10 degrees C was used to assess the combined effect of NaCl (0.5-4.5% w/v), pH (5.5-6.5) and atmosphere (10% H2:90% N2, 5% CO2:10% H2:85% N2, or 100% CO2) in buffered peptone, yeast, glucose, starch broth with an Eh of approximately -350 mV. Under all atmospheres growth tended to be slower as the concentration of NaCl increased and with NaCl combined with pH levels below 6.0. Of the atmospheres tested, growth occurred at a slower rate and over a narrower range of conditions when C. botulinum was exposed to 100% CO2. This effect was enhanced when the incubation temperature was 5 degrees C. The results indicate that while CO2 decreased C. botulinum growth at chill temperatures, prevention of growth also depended on the NaCl concentration and the pH of the medium.

Application of the Bigelow (z-value) model and histamine detection to determine the time and temperature required to eliminate Morganella morganii from seafood.

In New Zealand, the product most frequently implicated in cases of scombroid or histamine poisoning is the hot-smoked fish, kahawai (Arripis trutta). A properly controlled heating step in the production of hot-smoked seafood could eliminate bacteria able to convert the amino acid histidine to histamine. In this study, we determined the core temperatures and times required during hot smoking of kahawai to eliminate histamine-forming bacteria and to ensure a final product that will not produce histamine if subsequent temperature abuse occurs. Morganella morganii strains previously isolated from portions of hot-smoked kahawai with elevated histamine levels were inoculated onto product to be tested. A variation of the Bigelow or z-value model was used to generate a thermal death time graph, where the production of histamine, in a heat-treated and subsequently temperature-abused sample, was scored as a positive value (growth) and the absence of histamine was scored as a negative value (no growth). From a line fitted to the data, calculated times for the elimination of histamine-forming bacteria at test temperatures of 58, 59, 60, 61, and 62 degrees C were estimated to be 15.27, 8.81, 4.79, 2.68, and 1.46 min, respectively, giving a z value of 3.85 degrees C. This approach to thermal death determination, based on the presence or absence of a bacterial metabolite, proved to be an efficient way to determine the thermal regime required to eliminate bacteria capable of converting histidine to histamine on kahawai.

Survival of Streptococcus pyogenes under stress and starvation.

The ability of Streptococcus pyogenes to enter a quiescent state, similar to the stationary phase of lab cultures, is believed to be an important factor in its ability to persist within the host and to subsequently cause disease. Using a model broth system, we determined that after entering the stationary phase, there was a 99.99% reduction in cell viability over a 4-day period, following which the cells appeared to enter a resistant starvation state where cell numbers remained constant over the subsequent 3-4 weeks. This starvation response was induced by carbon or phosphorous limitation, but not by nitrogen limitation in the form of amino acids where cells became non-culturable after 4 days. Amino acid utilization in the absence of a carbon source may be an essential factor for the long-term survival of this bacterium in the stationary phase. Early stationary phase cells showed a greater resistance to oxidative and pH stress compared to 24-h-starved cultures. There was evidence for the formation of a viable but non-culturable state as indicated by a comparison of the numbers of cells with a functional membrane potential (rhodamine 123) against culturable cells on either Todd Hewitt broth agar or sheep blood agar. Long-term survival of S. pyogenes was dependent on both cell wall and protein synthesis, suggesting that starving cultures are a dynamic cell population.

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 degrees 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 degrees 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 degrees 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 degrees 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 degrees C showed an apparent increase in heat resistance after exposure to sea water at 20.8 degrees C for 7 d (D58 = 2.64 min) compared with before exposure (D58 = 1.24). This increase in thermal resistance was not apparent at temperatures greater than 63 degrees 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 degrees C).

Thermal death times of Hafnia alvei cells in a model suspension and in artificially contaminated hot-smoked kahawai (Arripis trutta).

In New Zealand the product most frequently implicated in cases of scombroid poisoning is hot-smoked kahawai (Arripis trutta). Using a Hafnia alvei strain, previously isolated from a portion of hot-smoked kahawai with a histamine level of 1,659.4 mg/kg, thermal death trials were carried out in a model suspension (0.1% peptone) at 54, 55, 56, 57, and 58 degrees C. From the linear regression line (R2 = 0.98) fitted to observed D values plotted against temperature, calculated D values for 54, 55, 56, 57, and 58 degrees C were estimated to be 0.63, 0.36, 0.20, 0.11, and 0.06 min, respectively, giving a z value of 4.14 degrees C. Thermal death trials were also carried out for H. alvei associated with hot-smoked kahawai at 54, 55, 55.5, 56, and 57 degrees C. From the linear regression line (R2 = 0.93) fitted to the data, calculated D values for 54, 55, 56, and 57 degrees C were estimated to be 1.42, 0.74, 0.38, and 0.20 min, respectively, giving a z value of 3.57 degrees C. Results indicate that hot smoking has the potential to eliminate H. alvei from seafood products.