Effect of high pressure, in combination with antilisterial agents, on the growth of Listeria monocytogenes during extended storage of cooked chicken.

A cocktail of Listeria monocytogenes strains was inoculated into cooked chicken (∼2.2 × 10³ CFU g⁻¹) which was then pressure-treated (600 MPa/2 min/20 °C) and stored for up to 105 days at 8 °C. In addition, sodium lactate (2% w/w) or a pressure-resistant Weissella viridescens strain, known to have antilisterial activity, were added to the meat prior to inoculation with the pathogen and pressure treatment, to investigate the effect on Listeria survival. Pressure treatment alone was not sufficient to eliminate all of the Listeria. Numbers of survivors were initially below the level of detection (50 CFU g⁻¹) but increased during storage to reach >108 CFU g⁻¹ by day 21. The presence of W. viridescens significantly extended the lag phase of any Listeria that survived the initial pressure treatment by ∼35 days, but numbers then increased to reach ∼107 CFU g⁻¹ by day 105. The addition of 2% sodium lactate in combination with pressure treatment was most effective at inhibiting the growth of L. monocytogenes and numbers remained below the limit of detection throughout the 105 day storage. The addition of antimicrobial agents, in combination with pressure, could be used to give additional food safety assurance without increasing pressure hold time.

Use of conductimetry to rapidly determine relative stress sensitivity in Salmonella isolates.

AIMS: To compare conventional plate counting and indirect conductimetry as techniques for ranking the resistance of Salmonella spp. to processing stressors. METHODS AND RESULTS: Forty Salmonella isolates were subjected to three separate stressors used in food processing; irradiation, heat and high hydrostatic pressure (HHP). Total viable counts (TVC) using conventional plate counts and time to detection (TTD) using indirect conductimetry were determined. A significant negative correlation between TVC and TTD was seen with irradiation (P < 0.01) and heat (P < 0.05) but not HHP. CONCLUSIONS: For a group of salmonellas, indirect conductimetry can rapidly determine a ranking of isolate sensitivity to irradiation and heat. However, for HHP, the results indicated that conventional plate counting alone cannot be used to determine sensitivity. SIGNIFICANCE AND IMPACT OF THE STUDY: The resistance of micro-organisms to processing systems must be ranked to allow the selection of appropriate isolates for process validation. TTD measurements allow rapid screening of salmonellas to rank isolates for resistance to irradiation and heat stress. However, following HHP, the TVC of survivors is independent of the time required for growth to a set cell density and therefore it cannot be used as the sole measure of relative stress resistance.

The combined effects of high pressure and nisin on germination and inactivation of Bacillus spores in milk.

AIMS: The aim of this work was to investigate the germination and inactivation of spores of Bacillus species in buffer and milk subjected to high pressure (HP) and nisin. METHODS AND RESULTS: Spores of Bacillus subtilis and Bacillus cereus suspended in milk or buffer were treated at 100 or 500 MPa at 40 degrees C with or without 500 IU ml(-1) of nisin. Treatment at 500 MPa resulted in high levels of germination (4 log units) of B. subtilis spores in both milk and buffer; this increased to >6 logs by applying a second cycle of pressure. Viability of B. subtilis spores in milk and buffer was reduced by 2.5 logs by cycled HP, while the addition of nisin (500 IU ml(-1)) prior to HP treatment resulted in log reductions of 5.7 and 5.9 in phosphate buffered saline and milk, respectively. Physical damage of spores of B. subtilis following HP was apparent using scanning electron microscopy. Treating four strains of B. cereus at 500 MPa for 5 min twice at 40 degrees C in the presence of 500 IU ml(-1) nisin proved less effective at inactivating the spores of these isolates compared with B. subtilis and some strain-to-strain variability was observed. CONCLUSIONS: Although high levels of germination of Bacillus spores could be achieved by combining HP and nisin, complete inactivation was not achieved using the aforementioned treatments. SIGNIFICANCE AND IMPACT OF THE STUDY: Combinations of HP treatment and nisin may be an appealing alternative to heat pasteurization of milk.

Effect of ante- and postmortem hide clipping on the microbiological quality and safety and ultimate pH value of beef carcasses in an EC-approved abattoir.

AIMS: Effect of ante- and postmortem hide clipping on the microbiological quality of beef carcasses. METHODS AND RESULTS: Bovine carcasses (362) were tested for indicator micro-organisms and the presence of pathogens. Prior to slaughter, hide cleanliness of each animal was categorized on a scale of 1-5 (clean to dirty). Lowest mean aerobic colony counts (ACC) (log(10) 3.0 CFU cm(-2)) came from carcasses where clipping had been performed in lairage, antemortem. ACC from animals clipped online (log(10) 3.2 CFU cm(-2)) were significantly higher (P < 0.05) than those clipped in lairage, but comparable to those carcasses from Category 1 and 2 animals. There were no significant differences in the detection of pathogens from any of the carcass groups. Ultimate pH values for carcasses from Category 3 and 4 animals showed clipping animals in lairage, as opposed to online, resulted in a small, but significant increase (P < 0.05) in pH value (mean pH 5.66 and 5.59, respectively). CONCLUSIONS: Hide clipping does not adversely affect microbiological quality of carcasses, although higher ultimate pH values indicate increases in antemortem stress. SIGNIFICANCE AND IMPACT OF THE STUDY: Hide clipping carcasses both ante- and postmortem appears to be an effective intervention to minimize transfer of hide microflora to carcasses during slaughtering operations. Online clipping offers advantages for animal welfare and improves safety for operatives.

Effect of high pressure and pasteurization on Mycobacterium avium ssp. paratuberculosis in milk.

AIMS: To determine the effect of high pressures alone and in conjunction with pasteurization on the viability of two strains of Mycobacterium avium ssp. paratuberculosis (Map). METHODS AND RESULTS: Map in a milk matrix was subjected to 400, 500 and 600 MPa with and without pasteurization (72 degrees C for 15 s) and plated onto Herrold's egg yolk medium (HEYM) and Middlebrook 7H10 (7H10) agar, both containing antibiotic supplements. Medium 7H10 was found to give a significantly (P < 0.001) better recovery than HEYM. A significantly greater (P < 0.001) reduction in viable numbers was observed using 500 MPa (mean log reduction of 6.52) compared with 400 MPa (mean log reduction of 2.56) and between 400 MPa and control (no applied pressure) for 10 min treatments. A treatment time of 10 min resulted in significantly (P < 0.001) fewer survivors than 5 min. Low numbers of survivors were still detected when pressure treatment at 400 and 600 MPa was combined with subsequent pasteurization. CONCLUSIONS: The use of high-pressure was effective in reducing viable numbers of Map but even when combined with pasteurization there were still survivors, albeit when high inoculum levels of Map were used. SIGNIFICANCE AND IMPACT OF THE STUDY: To the authors' knowledge the work reported here represents the first study of the efficacy of high-pressure treatments alone and in combination with pasteurization to kill Map. The results indicate that further research is warranted before more commercial-scale studies are commissioned.

Evaluation of the use of conductimetry for the rapid and precise measurement of Salmonella spp. growth rates.

The growth rates of 14 Salmonella serovars in tryptone soy broth plus yeast extract (TSBYE) were estimated using conventional plating techniques and indirect conductimetry using a Don Whitley RABIT system. Both methods gave identical results for the maximum specific growth rate (mumax) P>0.05. However, using the conductimetric method, mumax for a single serovar was determined in less than 7 h, whereas the conventional method required an additional 24 h. In addition, the conductimetric method was considerably more precise, much less labour-intensive and required the use of considerably less consumables. Using conductimetry, a trained operator could accurately determine mumax for 24 serovars in 3 working days, but only one serovar using the conventional plate counting technique. Hence, the use of conductimetry can markedly increase the precision and accuracy of mumax determinations by allowing a very significant increase in the number of results obtained and in their precision. The data generated will allow the development of better mathematical growth models. The method can also be used to compare growth media and conditions and hence rapidly optimise detection protocols for this pathogen.

Denervation produces different single fiber phenotypes in fast- and slow-twitch hindlimb muscles of the rat.

Using a single, mechanically skinned fiber approach, we tested the hypothesis that denervation (0 to 50 days) of skeletal muscles that do not overlap in fiber type composition [extensor digitorum longus (EDL) and soleus (SOL) muscles of Long-Evans hooded rats] leads to development of different fiber phenotypes. Denervation (50 day) was accompanied by 1) a marked increase in the proportion of hybrid IIB/D fibers (EDL) and I/IIA fibers (SOL) from 30% to >75% in both muscles, and a corresponding decrease in the proportion of pure fibers expressing only one myosin heavy chain (MHC) isoform; 2) complex muscle- and fiber-type specific changes in sarcoplasmic reticulum Ca(2+)-loading level at physiological pCa approximately 7.1, with EDL fibers displaying more consistent changes than SOL fibers; 3) decrease by approximately 50% in specific force of all fiber types; 4) decrease in sensitivity to Ca(2+), particularly for SOL fibers (by approximately 40%); 5) decrease in the maximum steepness of the force-pCa curves, particularly for the hybrid I/IIA SOL fibers (by approximately 35%); and 6) increased occurrence of biphasic behavior with respect to Sr(2+) activation in SOL fibers, indicating the presence of both slow and fast troponin C isoforms. No fiber types common to the two muscles were detected at any time points (day 7, 21, and 50) after denervation. The results provide strong evidence that not only neural factors, but also the intrinsic properties of a muscle fiber, influence the structural and functional properties of a particular muscle cell and explain important functional changes induced by denervation at both whole muscle and single cell levels.

Comparison of 40 Salmonella enterica serovars injured by thermal, high-pressure and irradiation stress.

AIM: To investigate and compare the inherent resistance of 40 Salmonella serovars to heat, irradiation and high-pressure stress. METHODS AND RESULTS: D10 values for each of the three stresses were calculated for four serovars, chosen as representatives from a catalogue of 40. Based on these results, conditions for each stress were defined, which produced, on average, a three-log reduction in viability. Heat stress (57 degrees C for 13 min), high-pressure stress (350 MPa for 10 min at 20 degrees C) and irradiation stress (1.5 kGy at 20 degrees C) were applied to all 40 serovars in the collection. Injury and loss of viability for all serovars were determined. CONCLUSIONS: Cluster analysis identified five groupings of isolates in terms of resistance to the applied stresses. The independent response of each isolate to all three stresses suggests that there is no relationship between resistances. SIGNIFICANCE AND IMPACT OF THE STUDY: Each serovar is inherently different. For modelling of real-life food preservation processing the most resistant isolates for that process should be chosen. The results also emphasize the importance of including multiple stress resistant strains when food preservation systems apply multiple stresses.

The ilio-marsupialis muscle in the dasyurid marsupial Sminthopsis douglasi: form, function and fibre-type profiles in females with and without suckling young.

The form, function and fibre-type profiles of the ilio-marsupialis muscles, branches of which insert on to the skin of the nipples and pouch, have been investigated in the small dasyurid marsupial Sminthopsis douglasi. Single fibres from the branches of muscles associated with unsuckled nipples in non-lactating females and with both unsuckled and suckled nipples at four stages during the 70-day suckling period were typed according to their sensitivity to the activators strontium (Sr(2+)) and calcium (Ca(2+)) into fast-twitch, slow-twitch and composite types. An unusual finding was the predominance of composite fibres in the resting state (unsuckled nipples). Changes in fibre-type composition were observed during the suckling period and these changes correlated with events in the development of the suckling young. Composite fibres declined during the suckling period and, at the stage when the young can no longer be accommodated in the pouch but must still be carried by the mother while she is foraging, an increase in fast-twitch fibres that are associated with dynamic muscular activity was seen. Later in the suckling period, when the mammary tissue is greatly enlarged but the mother does not carry the young while out feeding, there was an increase in the proportion of slow-twitch (fatigue-resistant) fibres. The high proportion of fast-twitch fibres present late in the suckling period may be associated with vibratory movements that result in the young relinquishing the nipples.

A study of the short-term effects of glucose 6-phosphate on the contractile activation properties of skinned single muscle fibres of the rat. Implications for solution design.

The present study highlights possible problems that can arise from the incorrect preparation of control and test solutions for use in Ca2+-activation experiments using single skinned skeletal muscle fibres and EGTA-based Ca2+ buffers. We show here, using glucose 6-phosphate (G6-P) as our "test" compound, that the Ca2+-activation properties of skinned single fibre segments from the extensor digitorum longus muscle of the rat are highly dependent on the form in which the G6-P is added and on the correct balancing of an appropriate anion in control solutions. Test solutions prepared by the direct addition of 10 mM monosodium G6-P salt to a set of control solutions of defined pCa resulted in significantly greater submaximal force responses than the corresponding controls. This is equivalent to an increase in the sensitivity of the contractile-regulatory system to Ca2+ (pCa50=-log10[Ca2+] that produces 50% of maximum force) by 0.19+/-0.01 pCa units. In contrast, addition of disodium G6-P to control solutions caused a slight reduction in the apparent sensitivity of the contractile apparatus to Ca2+ by 0.04+/-0.01 pCa units (P<0.01). Rather than being indicative of the effects of G6-P on the contractile apparatus, these opposing effects are due to differences between test and control solutions with respect to pH and Na+ concentration brought about by the G6-P salts. When all ionic species were carefully balanced, 10 mM G6-P was found to have only a small sensitizing effect on Ca2+-activation properties compared to control, without affecting the maximum Ca2+-activated force response. Our findings highlight the often-overlooked need for careful balancing of the ionic composition in control and test solutions when examining the true effects of different compounds on the Ca2+-activation characteristics of single skinned muscle fibre preparations.

The effect of growth stage and growth temperature on high hydrostatic pressure inactivation of some psychrotrophic bacteria in milk.

The effect of high hydrostatic pressure on the survival of the psychrotrophic organisms Listeria monocytogenes, Bacillus cereus, and Pseudomonas fluorescens was investigated in ultrahigh-temperature milk. Variation in pressure resistance between two strains of each organism were studied. The effect of growth stage (exponential and stationary phase), growth temperature (8 and 30 degrees C) on pressure resistance, and sublethal pressure injury were investigated. Exponential-phase cells were significantly less resistant to pressure than stationary-phase cells for all of the three species studied (P < 0.05). Growth temperature was found to have a significant effect at the two growth stages studied. Exponential cells grown at 8 degrees C were more resistant than those grown at 30 degrees C, but for stationary-phase cells the reverse was true. B. cereus stationary-phase cells grown at 30 degrees C were the most pressure resistant studied. L. monocytogenes showed the most sublethal damage compared to B. cereus and P. fluorescens. B. cereus spores were more resistant to pressure than vegetative cells. Pressure treatment at 400 MPa for 25 min at 30 degrees C gave a 0.45-log inactivation. Pressure treatment at 8 degrees C induced significantly less spore germination than at 30 degrees C. This study indicates the importance of the history of a bacterial culture prior to pressure treatment and that bacterial spores require more severe pressure treatments, probably in combination with other preservation techniques, to ensure inactivation.

Changes in the Ca2+-activation characteristics of demembranated rat single muscle fibres after prolonged incubation at room temperature in the presence and absence of DTT and protease inhibitors.

Prolonged incubation (24 h) of chemically skinned rat muscle preparations in rigor solutions at room temperature and in the absence of reducing agents and protease inhibitors modified the Ca2+-activation characteristics of the contractile machinery. In the absence of reducing agents and protease inhibitors, the contraction threshold for incubated fibres was shifted to lower Ca2+ concentrations and the steepness of the steady-state force-pCa (-log10)[Ca2+]) curve was decreased compared to that of control muscle fibres. Mean myosin ATPase activity under these conditions was significantly lowered by a factor of 2.7. Fibres incubated in the presence of 10 mM dithiothreitol (DTT) and protease inhibitors (100 microM pepstatin A/200 microM leupeptin) produced a maximum Ca2+-activated force per cross-sectional area that compared favourably with that of freshly dissected muscle fibres and there were no changes in the other contractile activation characteristics. Intermediate responses were obtained when fibres were incubated in the presence of either DTT or protease inhibitors. MgATPase activities of incubated preparations increased significantly following the addition of protease inhibitors and/or DTT to the incubation medium. Taken together, these results suggest that in the presence of DTT and protease inhibitors, most contractile properties are maintained at levels seen in fresh mechanically skinned fibres. The extended viability of this preparation and its closely related properties with fresh muscle fibres make it a useful model for experiments requiring longer term incubations with biological agents.

High pressure processing of foods for microbiological safety and quality (a short review).

Consumers are demanding foods that are "natural", of good nutritional and sensory quality, free from chemical preservatives, microbiologically safe and with extended shelf-life. High pressure processing can, potentially, meet these criteria. Recent advances in equipment design now allow foods to be processed up to 900 MegaPascals (130,000 psi). However, further work is required to more fully understand the factors that can affect the response of microorganisms, including pathogens, to pressure so that treatments can be optimised and microbiological safety can be assured. This paper describes how the pressure resistance of microorganisms can vary depending on factors such as species, strain, stage of growth and food composition. Strategies for overcoming the problem of pressure resistance will be discussed, for example the use of pressure cycling and the combination of pressure with mild heat. The current commercial uses of high pressure to preserve foods will be reported and potential applications will also be discussed.

Ca2+-activation characteristics of single fibres from chemically skinned rat muscle incubated with glucose-6-phosphate.

In this study we examined the effects of 3-24 h of incubation of chemically skinned rat fast-twitch muscle with the glycolytic metabolite glucose 6-phosphate (G6-P) on the contractile properties and myosin ATPase activity in single muscle fibres, and on the carbohydrate content of myosin heavy chains (MHCs). Exposure of the permeabilised muscle to 10 mM G6-P for 24 h at 22+/-1 degrees C in a rigor solution containing protease inhibitors and a reducing agent (dithiothreitol, DTT) significantly decreased maximum Ca(2+)-activated force output by 31%, lowered the Ca2+ threshold for contraction by 0.1 pCa units and produced shallower force-pCa curves compared with controls. Furthermore, under these conditions, G6-P-treated muscle displayed lower myofibrillar MgATPase activity and a markedly higher carbohydrate content of MHCs, as identified with an immunoblot protocol for glycoprotein detection. Shorter incubations under the same conditions or 24-h incubations with 5 mM G6-P generally resulted in smaller changes in the contractile activation parameters. These findings suggest that reducing sugars acting as metabolic intermediates in the glycolytic pathway can have important non-energy-related effects on the contractile activation characteristics of mammalian skeletal muscle. These effects are consistent with the glycation of muscle proteins, in particular that of the MHC.

Phylogenetic characterization of a novel radiation-resistant bacterium from irradiated pork: description of Hymenobacter actinosclerus sp. nov.

A phylogenetic analysis was performed on a red-pigmented, radiation-resistant, Gram-negative, rod-shaped organism originating from irradiated pork. Comparative 16S rRNA gene sequencing showed the bacterium was a member of the Cytophaga-Flavobacterium-Bacteroides line of descent and represents a new subline within the genus Hymenobacter. A new species, Hymenobacter actinosclerus, is described for this novel radiation-resistant bacterium. The type strain of Hymenobacter actinosclerus is CCUG 39621T.

Survival of Escherichia coli O157:H7 during storage in pressure-treated orange juice.

The effect of a high-pressure treatment on the survival of a pressure-resistant strain of Escherichia coli O157:H7 (NCTC 12079) in orange juice during storage at 3 degrees C was investigated over the pH range of 3.4 to 5.0. The pH of shelf-stable orange juice was adjusted to 3.4, 3.6, 3.9, 4.5, and 5.0 and inoculated with 10(8) CFU ml(-1) of E. coli O157:H7. The orange juice was then pressure treated at 400 MPa for 1 min at 10 degrees C or was held at ambient pressure (as a control). Surviving E. coli O157:H7 cells were enumerated at 1-day intervals during a storage period of 25 days at 3 degrees C. Survival of E. coli O157:H7 during storage was dependent on the pH of the orange juice. The application of high pressure prior to storage significantly increased the susceptibility of E. coli O157:H7 to high acidity. For example, after pressure treatment, the time required for a 5-log decrease in cell numbers was reduced from 13 to 3 days at pH 3.4, from 16 to 6 days at pH 3.6, and from >25 to 8 days at pH 3.9. It is evident that the use of high-pressure processing of orange juice in order to increase the juice's shelf-life and to inactivate pathogens has the added advantage that it sensitizes E. coli O157:H7 to the high acid conditions found in orange juice, which results in the survival of significantly fewer E. coli O157:H7 during subsequent refrigerated storage.

Inactivation of Escherichia coli O157:H7 in orange juice using a combination of high pressure and mild heat.

The effect of high pressure on the survival of a pressure-resistant strain of Escherichia coli O157:H7 (NCTC 12079) in orange juice was investigated over the pH range 3.4 to 5.0. The pH of commercial, sterile orange juice was adjusted to 3.4, 3.6, 3.9, 4.5, or 5.0. The juice was then inoculated with 10(8) CFU ml(-1) of E. coli O157:H7. The inoculated orange juice was subjected to pressure treatments of 400, 500, or 550 MPa at 20 degrees C or 30 degrees C to determine the conditions that would give a 6-log10 inactivation of E. coli O157:H7. A pressure treatment of 550 MPa for 5 min at 20 degrees C produced this level of kill at pH 3.4, 3.6, 3.9, and 4.5 but not at pH 5.0. Combining pressure treatment with mild heat (30 degrees C) did result in a 6-log10 inactivation at pH 5.0. Thus, the processing conditions (temperature and time) must be considered when pressure-treating orange juice to ensure microbiological safety.

The combined effect of high hydrostatic pressure and mild heat on inactivation of pathogens in milk and poultry.

The combined effects of high hydrostatic pressure and heat on the inactivation of Escherichia coli O157:H7 NCTC 12079 and Staphylococcus aureus NCTC 10652 in poultry meat and ultra-high-temperature-treated (UHT) milk were investigated. The to have a significant effect on the survival of the pathogens during treatment. For E. coli O157:H7, a 15-min treatment of 400 MPa at 50 degrees C resulted in approximately a 6.0-log10 reduction in CFU/g in poultry meat and a 5.0-log10 reduction in UHT milk; however, a < 1-log10 reduction was achieved with either treatment alone. In contrast, for S. aureus, a 15-min treatment of 500 MPa at 50 degrees C was required to achieve a 5.0-log10 reduction in poultry meat and a 6.0-log10 reduction in UHT milk. As before, a < 1-log10 reduction in numbers was achieved with either treatment alone. The pressure-temperature inactivation curves of each organism, in each substrate, were fitted using the Gompertz equation. Polynomial expression derived from the Gompertz variables were used to devise simple models which predicted the inactivation of each pathogen at various pressure-temperature combinations. Thus, a number of different pressure-temperature conditions could be chosen to achieve a desired inactivation level. The use of such models will provide flexibility in selecting optimum pressure conditions without compromising microbiological safety.

Basis for late rise in fura 2 R signal reporting [Ca2+]i during relaxation in intact rat ventricular trabeculae.

Intact rat ventricular trabeculae were injected with the salt form of fura 2, and the fura 2 ratio signal (R) was used to report intracellular Ca2+ concentration ([Ca2+]i). The fixed end relaxation phase of a twitch is associated with a slowing of the decay of the R signal, or even a reversal, to form a distinct bump, indicating a transient rise in [Ca2+]i. The bump is most prominent at 30 degrees C, and motion artifact is not its cause. Increasing doses of 2,3-butanedione monoxime caused progressive attenuation of the twitch and bump. Increasing the bathing Ca2+ concentration potentiated the twitch and enhanced the bump. Imposed muscle shortening during relaxation caused a much quicker force decline, and this led to the appearance of a much more prominent associated bump. The amplitude of the bump depends on the amplitude of twitch force and the rate of relaxation. These findings can be explained, as in skeletal muscle, by making cross-bridge attachment and Ca2+ binding to troponin C strongly cooperative; therefore, the bump during fast relaxation is produced by a reversal of this cooperatively, leading to rapid dissociation of Ca2+ from troponin C into the myoplasm.