Use of the Weibull model for lactococcal bacteriophage inactivation by high hydrostatic pressure

Four lactococcal bacteriophages (phiLl6-2, phiLl35-6, phiLd66-36 and phiLd67-42) in M17 broth were pressurized at 300 and 350 MPa at room temperature and their survival curves were determined at various time intervals. Tailing (monotonic upward concavity) was observed in all survival curves. The resulting non-linear semi-logarithmic survival curves were described by the Weibull model and goodness of fit of this model was investigated. Regression coefficients (R2), root mean square error (RMSE), residual and correlation plots strongly suggested that Weibull model produced a better fit to the data than the traditional linear model. Hazard plots suggested that the Weibull model was fully appropriate for the data being analyzed. These results have confirmed that the Weibull model, which is mostly utilized to describe the inactivation of bacterial cells or spores by heat and pressure, could be successfully used in describing the lactococcal bacteriophage inactivation by high hydrostatic pressure.     

Bacterial inactivation by high-pressure homogenisation and high hydrostatic pressure

The resistance of five gram-positive bacteria, Enterococcus faecalis, Staphylococcus aureus, Lactobacillus plantarum, Listeria innocua and Leuconostoc dextranicum, and six gram-negative bacteria, Salmonella enterica serovar typhimurium, Shigella flexneri, Yersinia enterocolitica, Pseudomonas fluorescens and two strains of Escherichia coli, to high-pressure homogenisation (100-300 MPa) and to high hydrostatic pressure (200-400 MPa) was compared in this study. Within the group of gram-positive bacteria and within the group of gram-negative bacteria, large differences were observed in resistance to high hydrostatic pressure, but not to high-pressure homogenisation. All gram-positive bacteria were more resistant than any of the gram-negative bacteria to high-pressure homogenisation, while in relative to high hydrostatic pressure resistance both groups overlapped. Within the group of gram-negative bacteria, there also existed another order in resistance to high-pressure homogenisation than to high hydrostatic pressure. Further it appears that the mutant E. coli LMM1010, which is resistant to high hydrostatic pressure is not more resistant to high-pressure homogenisation than its parental strain MG1655. The preceding observations indicate a different response of the test bacteria to high-pressure homogenisation compared to high hydrostatic pressure treatment, which suggests that the underlying inactivation mechanisms for both techniques are different. Further, no sublethal injury could be observed upon high-pressure homogenisation of Y. enterocolitica and S. aureus cell population by using low pH (5.5 7), NaCl (0 6%) or SDS (0-100 mg/l) as selective components in the plating medium. Finally, it was observed that successive rounds of high-pressure homogenisation have an additive effect on viability reduction of Y. enterocolitica and S. aureus.     

Activation and inactivation of Talaromyces macrosporus ascospores by high hydrostatic pressure

The effect of high hydrostatic pressure treatment (with pressures of up to 700 MPa) on Talaromyces macrosporus ascospores was investigated. At 20 degrees C, pressures of > or = 200 MPa induced the activation and germination of dormant ascospores, as indicated by increased colony counts for ascospore suspensions after pressure treatment and the appearance of germination vesicles and tubes. Pressures of > 400 MPa additionally sensitized the ascospores to subsequent heat treatment. At pressures of > 500 MPa, activation occurred in a few minutes but was followed by inactivation with longer exposure. However, even with the most extreme pressure treatment, a fraction of the ascospore population appeared to resist both activation and inactivation, and the maximal achievable reduction of ascospores was on the order of 3.0 log10 units. Pressure-induced ascospore activation at 400 MPa was temperature dependent, with minimum activation at 30 to 50 degrees C and > or = 10-fold higher activation levels at 10 to 20 degrees C and at 60 degrees C, but it was not particularly pH dependent over a pH range of 3.0 to 6.0. Pressure inactivation at 600 MPa, in contrast, was pH dependent, with the inactivation level being 10-fold higher at pH 6.0 than at pH 3.0. Observation of pressure-treated and subsequently dried spores with the use of light and scanning electron microscopy revealed a collapse of the spore structure, indicating a loss of the spore wall barrier properties. Finally, pressure treatment sensitized T. macrosporus ascospores to cell wall lytic enzymes.     

Kinetic analysis of E. coli inactivation by high hydrostatic pressure with salts

Inactivation of E. coli by high hydrostatic pressure (250 to 400 MPa) with salts was investigated based on kinetic analysis. At concentrations from 0.074 to 0.145 M and from 0.240 to 0.290 M, both the absolute activation volumes and the preexponential factors were similar in KCl, NaCl, and LiCl solutions, suggesting that pressure inactivation is not salt-specific. On the other hand, in the intermediate salt-concentration range of 0.145 to 0.240 M, inactivation kinetics in the presence of the Na(+) and K(+) differed significantly from those in the presence of Li(+) (P < 0.05). In this concentration range, effect of salt stress and osmotic stress differed significantly from those in concentrations below 0.145 M or above 0.240 M. The cellular response to pressure varies with salt type and salt concentration. These novel findings provide important clues to distinguish between salt stress and osmotic stress in the inactivation of E. coli.     

Model for Listeria monocytogenes inactivation on dry-cured ham by high hydrostatic pressure processing

The aim of the work was to develop and validate a model of the inactivation of Listeria monocytogenes on dry-cured ham by high hydrostatic pressure (HHP) processing, as a function of the technological parameters: intensity, length and fluid temperature. Dry-cured ham inoculated with L. monocytogenes was treated at different HHP conditions (at 347-852 MPa; for 2.3 to 15.75 min; at 7.6 to 24.4 °C) following a central composite design. Bacterial inactivation was assessed in terms of logarithmic reductions of L. monocytogenes counts on selective media. According to the best fitting and most significant polynomial equation, pressure and time were the most important factors determining the inactivation extent. The significance of the quadratic term of pressure and time indicated that little effect was observed below 450 MPa, whereas holding time longer than 10 min did not result in a meaningful reduction of L. monocytogenes counts. Temperature did not show significant influence at the range assayed. The model was validated with results obtained from further experiments and bibliographical data within the range of the experimental domain. The accuracy factor and bias factor were within the proposed acceptable values indicating the suitability of the model for predictive purposes, such as prediction of the process criteria to meet the Food Safety Objectives. The results of this work may help food processors to select optimum processing conditions of HHP.     

Multi-pulsed high hydrostatic pressure treatment for inactivation and injury of Escherichia coli

Escherichia coli cells in peptone water were pressurized at 300?MPa at ambient temperature with no holding time (pulse series) and with a total holding duration of 300?s for single- (300?s?×?1 pulse) and multi-pulsed (150?s?×?2 pulses, 100?s?×?3 pulses, 75?s?×?4 pulses, 60?s?×?5 pulses, 50?s?×?6 pulses and 30?s?×?10 pulses) high hydrostatic pressure (HHP) treatments. Multi-pulsed HHP treatment with no holding time indicated that as the pulse number increased the number of inactivated and injured cells also increased. Holding time had significant effect on the inactivation of E. coli. There was low inactivation difference between single- and multi-pulsed HHP treatments with holding time. Escherichia coli cells showed at least 1.6 log₁₀ more reduction on selective medium than the non-selective medium indicating that more than 95?% of the survivors severely injured for both single- and multi-pulsed treatments with holding time. Although the inactivation difference was low between single- and multi-pulsed HHP treatments, storage at 4?°C revealed that there was less recovery from injury for multi-pulsed HHP treatment.     

Inactivation of avian influenza virus by heat and high hydrostatic pressure

Avian influenza viruses threaten the life of domestic terrestrial poultry and contaminate poultry meat and eggs. Recently, these viruses rarely infected humans but had a high mortality rate in Southeast Asia, the Middle East, and Egypt. Thereby, these viruses caused high economic costs for production of poultry and health protection. We inactivated a highly pathogenic avian influenza A virus of subtype H7N7 in cell culture medium and chicken meat by heat and high hydrostatic pressure. Because heat and pressure inactivation curves of the H7N7 virus showed deviations from first-order kinetics, a reaction order of 1.1 had to be selected. A mathematical inactivation model has been developed that is valid between 10 and 60 degrees C and up to 500 MPa, allowing the prediction of the reduction in virus titer in response to pressure, temperature, and treatment time. Incubation at 63 degrees C for 2 min and 500 MPa at 15 degrees C for 15 s inactivated more than 10(5) PFU/ml, respectively. Thus, we suggest high-pressure treatment of poultry and its products to avoid the possible health threat by highly pathogenic avian influenza viruses.     

超高压处理对泡豇豆杀菌效果的影响

为了探求超高压杀菌在四川泡菜工业中的应用,提高其微生物安全,以泡豇豆为供试材料,研究了泡豇豆含盐量、处理压力、处理时间对超高压杀菌效果的影响,并考察了超高压处理对泡豇豆保藏性能的影响。实验结果表明:含盐量为4.2%的泡豇豆超高压杀菌效果优于含盐量6.7%,超高压技术更适用于低盐泡菜的杀菌;压力越大、处理时间越长,微生物致死率越高,但压力较时间对杀菌效果的影响更大;对样品分别采用300、400MPa处理30min,菌落总数降低的对数为5.45、5.52,且均无霉菌和酵母菌检出;300～400MPa处理可以使大肠菌群数有效得到降低,且能有效提高泡豇豆的保藏性能。     

Environmental factors influencing the inactivation of Cronobacter sakazakii by high hydrostatic pressure

The effect of High Hydrostatic Pressure (HHP) on the survival of Cronobacter sakazakii was investigated. Deviations from linearity were found on the survival curves and the Mafart equation accurately described the kinetics of inactivation. Comparisons between strains and treatments were made based on the time needed for a 5-log₁₀ reduction in viable count. The ability of C. sakazakii to tolerate high pressure was strain-dependent with a 26-fold difference in resistance among four strains tested. Pressure resistance was greatest in the stationary growth phase and at the highest growth temperatures tested (30 and 37 °C). Cells treated in neutral pH buffer were 5-fold more resistant than those treated at pH 4.0, and 8-fold more sensitive than those treated in buffer with sucrose added (a_w = 0.98). Pressure resistance data obtained in buffer at the appropriate pH adequately estimated the resistance of C. sakazakii in chicken and vegetables soups. In contrast, a significant protective effect against high pressure was conferred by rehydrated powdered milk. As expected, treatment efficacy improved as pressure increased. z values of 112, 136 and 156 MPa were obtained for pH 4.0, pH 7.0 and a_w = 0.98 buffers, respectively. Cells with sublethal injury to their outer and cytoplasmic membranes were detected after HHP under all the conditions tested. The lower resistance of C. sakazakii cells when treated in media of pH 4.0 seemed to be due to a decreased barostability of the bacterial envelopes. Conversely, the higher resistance displayed in media of reduced water activity may relate to a higher stability of bacterial envelopes.     

Effect of high hydrostatic pressure and temperature on carrot peroxidase inactivation

The combined effects of pressure and temperature on the activity of carrot peroxidase (POD) were investigated in the pressure range 0.1–600 MPa and the temperature range 25–45 °C. At lower pressures (<396 MPa), carrot POD stability increased compared to unpressurized samples. Inactivation of 91% was obtained at 600 MPa and 45 °C. High hydrostatic pressure (HHP) combined with temperature treatment enhanced the inactivation of carrot POD. Regeneration of POD activity with the combined HHP and temperature treatments followed first order kinetics at 25, 35 and 40 °C. Regeneration was not observed at 506 MPa and 45 °C. HHP had no significant effect on the loss of vitamin C or on protein content. HHP combined with mild heat treatment was found to be better than the thermal treatment at high temperatures for inactivation of POD in carrot processing.     

超高压对双孢蘑菇的杀菌效果和动力学的研究

以细菌总数、大肠菌群、酵母菌和霉菌数为对象,研究了超高压(High hydrostatic pressure,HHP)处理对双孢蘑菇(Agaricus bisporus)的杀菌效果和杀菌动力学。双孢蘑菇在300、400、500、600MPa压力下,室温下分别用HHP处理2.5～25min。结果表明:随压力的升高和时间的延长,杀菌效果增强;霉菌、酵母对压力较为敏感,400MPa处理2.5min可将其全部杀死;300MPa处理2.5min可完全杀灭双孢蘑菇中的大肠菌群。应用Weibull模型对不同处理条件下双孢蘑菇的杀菌效果进行拟合,拟合动力学曲线的决定系数R2均大于0.97,拟合效果较好。提出了双孢蘑菇的HHP杀菌的最优杀菌工艺参数,即600MPa处理5min,该条件即可以有效杀灭双孢蘑菇中的微生物。     

Multi-pulsed high hydrostatic pressure treatment for inactivation and injury of Escherichia coli

Escherichia coli cells in peptone water were pressurized at 300 MPa at ambient temperature with no holding time (pulse series) and with a total holding duration of 300 s for single- (300 s × 1 pulse) and multi-pulsed (150 s × 2 pulses, 100 s × 3 pulses, 75 s × 4 pulses, 60 s × 5 pulses, 50 s × 6 pulses and 30 s × 10 pulses) high hydrostatic pressure (HHP) treatments. Multi-pulsed HHP treatment with no holding time indicated that as the pulse number increased the number of inactivated and injured cells also increased. Holding time had significant effect on the inactivation of E. coli. There was low inactivation difference between single- and multi-pulsed HHP treatments with holding time. Escherichia coli cells showed at least 1.6 log₁₀ more reduction on selective medium than the non-selective medium indicating that more than 95 % of the survivors severely injured for both single- and multi-pulsed treatments with holding time. Although the inactivation difference was low between single- and multi-pulsed HHP treatments, storage at 4 °C revealed that there was less recovery from injury for multi-pulsed HHP treatment.     

Effect of initial concentration of bacterial suspensions on their inactivation by high hydrostatic pressure

The effects of initial concentration [10⁴–10⁹ colony forming units (CFU) mL⁻¹] on the inactivation of vegetative cell suspensions ( Escherichia coli ) and spore suspensions ( Bacillus subtilis ) by hydrostatic pressure treatment were investigated. The inactivation rates of E. coli and B. subtilis decreased as the initial concentration of cell and spore suspensions increased. In the practical application of hydrostatic pressure treatment, it was considered that the initial concentration of the bacteria suspensions should be as low as possible.     

A kinetic study on inactivation of tilapia myosin Ca-ATPase induced by high hydrostatic pressure

Tilapia myosin (2.5 mg/ml) was treated by hydrostatic pressure (50–300 MPa) for 0–60 min to determine the inactivation kinetics of myosin Ca-ATPase. The process of the pressure-induced inactivation of myosin Ca-ATPase included two steps: the first one was an instantaneous pressure-induced inactivation, and the degrees of lost activities, called instantaneous pressure kill (IPK) values, increased with elevated pressure. The second one, the logarithm of residual activity of myosin ATPase, decreased smoothly during each pressure treatment for 10–60 min. However, D values (the time needed for 90% loss of the activity during a treatment at the same pressure) of Ca-ATPase decreased about 50% with per 50 MPa increase. In this study, 150 MPa was the pressure level that caused apparent myosin denaturation and the typical network structure formation with beyond 50% decrease of myosin Ca-ATPase activity.     

超高压杀菌效果及机制研究进展

超高压作为一种新型的食品非热加工技术, 处理过程温度低、对食品营养成分破坏小,能在有效杀菌的同时显著提升加工食品品质,是未来食品加工技术发展的热点方向。近年来,超高压技术被广泛应用于食品加工,并在国内外实现了商业化应用。杀菌作为超高压加工食品过程中最重要的环节,是保证食品安全、延长产品货架期的关键点,因此一直是本领域研究的重点。本文介绍了超高压技术的设备和作用原理, 总结了超高压或超高压联合其他手段对微生物营养体、细菌芽孢、真菌孢子及病毒的杀灭效果和杀菌机制, 归纳了超高压杀菌中存在的杀菌机制不清、缺乏杀菌指示菌以及深休眠芽孢等问题, 以期为进一步完善超高压杀菌理论、推动超高压技术在食品加工中的产业化应用指明方向。     

Food processing by high hydrostatic pressure

The use of high hydrostatic pressures (HHP) for food processing is finding increased application within the food industry. One of the advantages of this technology is that because it does not use heat, sensory, and nutritional attributes of the product remain virtually unaffected, thus yielding products with better quality than those processed traditional methods. HHP have the ability to inactivate microorganisms as well as enzymes responsible for shortening the life of a product. In addition to lengthening the shelf-life of food products, HHP can modify functional properties of components such as proteins, which in turn can lead to the development of new products. Equipment for large-scale production of HHP processed products are commercially available nowadays. Guacamole, sliced ham, oysters, and fruit juices are some of the products currently available on the market. HHP technology is one of the most promising nonthermal processes.     

Mechanisms of Bacillus subtilis spore inactivation by single- and multi-pulse high hydrostatic pressure (MP-HHP)

Herein we investigate the effect of multi-pulse high hydrostatic pressure (MP-HHP) on the inactivation of Bacillus subtilis spores. B. subtilis spores were subjected to MP-HHP under pressures at 200–500 MPa at temperatures of 40 and 60 °C with 3 pulses (holding time of 3 min) with a total processing time of 10 min and compared it with a single pressurization (S-HHP).Mechanism of spore inactivation by S- or MP-HHP was explored by assessing germination by heat shock treatment, spore susceptibility to lysozyme and hydrogen peroxide (H₂O₂), release of dipicolinic acid (DPA), and the permeability of inner membrane and cortex. Our results presented the highest spore inactivation (5.8 log reduction), when MP-HHP was applied under the highest temperature and pressure. The increased inactivation appears to be largely due to mechanical disruption of spore coat and inner and outer membranes, as evidenced by DPA release, increased susceptibility to lysozyme and H₂O₂ (indicative of breakage of disulfide bonds in the spore coat), and membrane permeability as assessed by spore staining and fluorescence microscopy. No differences were seen in germination between MP-HHP and S-HHP. There was no evidence of any loss of cortex lytic enzymes or degradation of small acid-soluble proteins (SASPs) during both MP-HHP and S-HHP treatments.     

Resistance of poliovirus to inactivation by high hydrostatic pressures

Suspensions of poliovirus, a typical enteric virus, were subjected to high hydrostatic pressures of 200 MPa for 15 min, 400 MPa for 15 min, 600, MPa for 15 min, and 600 MPa for 1 h. Suspensions of respiratory adenovirus (a dissimilar virus type) were treated likewise for comparison. Whereas adenoviruses were inactivated at pressures of 400 MPa and above, poliovirus showed no statistically significant inactivation by hydrostatic pressures up to 600 MPa for 1 h. This finding may have implications for the use of high pressure in food processing, as poliovirus has physical similarities to significant foodborne pathogenic enteric viral agents such as hepatitis A virus.     

Lytic and nonlytic mechanism of inactivation of gram-positive bacteria by lysozyme under atmospheric and high hydrostatic pressure

A different behavior was observed in three gram-positive bacteria exposed to hen egg white lysozyme by plate counts and phase-contrast microscopy. The inactivation of Lactobacillus johnsonii was accompanied by spheroplast formation, which is an indication of peptidoglycan hydrolysis. Staphylococcus aureus was resistant to lysozyme and showed no signs of peptidoglycan hydrolysis, and Listeria innocua was inactivated and showed indications of cell leakage but not of peptidoglycan hydrolysis. Under high hydrostatic pressure, S. aureus also became sensitive to lysozyme but did not form spheroplasts and was not lysed. These results suggested the existence of a nonlytic mechanism of bactericidal action of lysozyme on the latter two bacteria, and this mechanism was further studied in L. innocua. Elimination of the enzymic activity of lysozyme by heat denaturation or reduction with beta-mercaptoethanol eliminated this bactericidal mechanism. By means of a LIVE/DEAD viability stain based on a membrane-impermeant fluorescent dye, the nonlytic mechanism was shown to involve membrane perturbation. In the absence of lysozyme, high-pressure treatment was shown to induce autolytic activity in S. aureus and L. innocua.