pH and solute concentration of suspension media affect the outcome of high hydrostatic pressure treatment of Listeria monocytogenes

The effect of pH and solute concentration of suspension media on high hydrostatic pressure (HHP) induced inactivation of Listeria monocytogenes (approximate 10(8) CFU/ml) was investigated by the using treatment between 300 MPa and 600 MPa at 25 degrees C for 10 min. The suspension media used in this study represented different concentrations (0.1% to 10%) of buffered peptone water (BPW) with an adjusted pH of 4 to 7. An increase in the concentration of BPW resulted in a decreased HHP-induced inactivation of L. monocytogenes that was dependent on the pH of the medium. HHP-treatment at 300 MPa showed no bactericidal effect at neutral pH regardless of the BPW concentration. When the pH of BPW (0.1% to 5%) was reduced to 4, L. monocytogenes was completely inactivated (more than an 8 log cycle reduction) with a HHP-treatment of at least 300 MPa. HHP-treatment above 400 MPa completely inactivated L. monocytogenes in a relatively dilute BPW (0.1% and 1%) with an adjusted pH below 6. While only a 2 log cycle reduction was observed in 10% BPW at the pH ranging from 5 to 7 after treatment with 600 MPa, L. monocytogenes in 10% BPW at pH 4 was completely inactivated. Even though a significant bactericidal effect of HHP-treatment was not observed when applied with a low pressure such as 300 MPa or suspended in higher BPW at neutral pH, a reduction of the pH greatly affected the HHP-induced inactivation of L. monocytogenes. These results indicated that information concerning the pH of food or media would greatly assist an optimization of HHP-treatment for the inactivation of bacteria.     

Progressive freeze concentration of whey protein–sucrose–salt mixtures

Progressive freeze concentration of whey protein solutions is evaluated. Since solutions in industry are more complex, the effect of the addition of sodium chloride and sucrose on the inclusion behaviour is studied as well. Using a progressive freeze concentrator solutions of whey protein and mixtures of whey protein and/or sucrose and/or sodium chloride were freeze concentrated. At an initial concentration of 4%(w/w), whey proteins were not included in the ice fraction. At higher concentrations the inclusions are caused by the increase in viscosity in the boundary layer, impeding mass transfer. The addition of sucrose caused a similar effect. Presence of sodium chloride causes inclusions through the occurrence of a zone where the solution is locally super-cooled and leads to the formation of dendritic ice which encapsulates pockets of solution in the ice layer. Mixtures of both sucrose and sodium chloride gave no additive effect on solute inclusion but just a concurrent effect.     

Combined high pressure and temperature induced lethal and sublethal injury of Lactococcus lactis--application of multivariate statistical analysis

It was the aim of this work to determine the combined effects of pressure, temperature, and co-solutes on Lactococcus lactis, and to detect correlations between culture-dependent and culture-independent methods for assessment of cellular viability and sublethal injury. Therefore, the pressure induced inactivation of L. lactis MG 1363 was investigated in buffer and in buffer with 1.5 M sucrose or 4 M NaCl at a pressure range of 0.1 to 500 MPa and a temperature range of 5 to 50 degrees C. The inactivation was characterised by viable cell counts, stress resistant cell counts, membrane integrity, metabolic activity, and the activity of the multi-drug-resistance transport enzyme LmrP. L. lactis was most resistant to pressure application at 20-30 degrees C. Sucrose protected towards inactivation at any temperature, NaCl provided protection at high temperatures only. By using Principal Component Analysis, correlations were detected between viable cell counts and metabolic activity as well as stress resistant cell counts and LmrP activity. In conclusion, the pressure-inactivation of L. lactis is strongly temperature dependent, baroprotection by sucrose occurs at any temperature but the baroprotective effects of NaCl is temperature dependent. Further on, a combination of two experimental methods fully describe lethal and sublethal injury of pressure treated cells. These simplification of data acquisition and model development facilitates the establishment of pressure processes in food technology.     

Use of phenolic compounds for sensitizing Listeria monocytogenes to high-pressure processing

Three Listeria monocytogenes strains (Scott A, OSY-8578, and OSY-328) that differ considerably in barotolerance were grown to stationary phase and suspended individually in phosphate buffer (pH 7.0). Twelve phenolic compounds, including commercially used food additives, were screened for the ability to sensitize L. monocytogenes to high-pressure processing (HPP). Each L. monocytogenes strain was exposed to each of the 12 phenolic compounds (100 ppm each) for 60 min; this was followed by a pressure treatment at 400 MPa for 5 min. Six phenolic compounds increased the efficacy of HPP against L. monocytogenes but tert-butylhydroquinone (TBHQ) was the most effective. The additives alone at 100 ppm were not lethal for L. monocytogenes. Subsequently, the three L. monocytogenes strains were exposed to TBHQ before or after pressure treatments at 400 or 500 MPa for 5 min. When TBHQ was added after the pressure treatment, the combined treatment was more lethal than was pressure alone. However, the lethality attributable to TBHQ was greater when the additive was applied before rather than after pressure treatment. The inactivation kinetics of the L. monocytogenes strains at 300, 500, and 700 MPa, in the presence or absence of TBHQ, was investigated. All survivor plots showed non-linear inactivation kinetics, but tailing behavior was most pronounced when HPP was used alone. Combinations of TBHQ and HPP eliminated tailing behavior when survivors were monitored by direct plating or an enrichment procedure. Pressure and phenolic additives are apparently a potent bactericidal combination against L. monocytogenes.     

Inactivation of barotolerant Listeria monocytogenes in sausage by combination of high-pressure processing and food-grade additives

Food-grade additives were used to enhance the efficacy of high-pressure processing (HPP) against barotolerant Listeria monocytogenes. Three strains of L. monocytogenes (Scott A, OSY-8578, and OSY-328) were compared for their sensitivity to HPP, nisin, tert-butylhydroquinone (TBHQ), and their combination. Inactivation of these strains was evaluated in 0.2 M sodium phosphate buffer (pH 7.0) and commercially sterile sausage. A cell suspension of L. monocytogenes in buffer (10(9) CFU/ml) was treated with TBHQ at 100 ppm, nisin at 100 IU/ml, HPP at 400 MPa for 5 min, and combinations of these treatments. Populations of strains Scott A, OSY-8578, and OSY-328 decreased 3.9, 2.7, and 1.3 log with HPP alone and 6.4, 5.2, and 1.9 log with the HPP-TBHQ combination, respectively. Commercially sterile sausage was inoculated with the three L. monocytogenes strains (10(6) to 10(7) CFU/g) and treated with selected combinations of TBHQ (100 to 300 ppm), nisin (100 and 200 ppm), and HPP (600 MPa, 28 degrees C, 5 min). Samples were enriched to detect the viability of the pathogen after the treatments. Most of the samples treated with nisin, TBHQ, or their combination were positive for L. monocytogenes. HPP alone resulted in a modest decrease in the number of positive samples. L. monocytogenes was not detected in any of the inoculated commercial sausage samples after treatment with HPP-TBHQ or HPP-TBHQ-nisin combinations. These results suggest that addition of TBHQ or TBHQ plus nisin to sausage followed by in-package pressurization is a promising method for producing Listeria-free ready-to-eat products.     

Baroprotective effect of increased solute concentrations on yeast and moulds during high pressure processing

The baroprotective effect of increasing solute concentrations on yeast cells and fungal conidia subjected to high pressure processing (HPP) was studied. Suspensions of yeast cells (Saccharomyces cerevisiae, Pichia anomala and Hanseniaspora uvarum) or fungal spores (Penicillium expansum, Fusarium oxysporum and Rhizopus stolonifer) in citrate phosphate buffer formulated with sucrose at 40, 50 and 60 °Brix, or with glycerol and NaCl at equivalent water activity (a_w) values (0.925, 0.903 and 0.866 a_w) were subjected to 600 MPa pressure for varying times, and then were enumerated by spread plate technique to assess survival. There was an increasing resistance to inactivation by high pressure with an increase in solute concentration. The two moulds with easily wetted spores, R. stolonifer and F. oxysporum, showed strongest resistance to HPP at 0.866 a_w. Differing responses to the three solutes were observed among the fungal species tested, indicating that the chemical nature of the solute may also be important in protecting yeasts and moulds during and after pressure treatment. Sucrose had a stronger baroprotective effect for S. cerevisiae than the other solutes, at two of the three investigated a_ws. For P. expansum at 0.903 a_w, NaCl gave the best protective effect. Scanning electron microscopy of HPP treated cells showed the protective effects of increased sucrose concentration. The results reported here have practical implications for the food industry in the application of HPP for production of fruit preparations or syrups, and should be taken in account in establishing efficient process design.

Industrial relevance

As high concentrations of sugar, salt and glycerol provide protection for yeasts and moulds during high pressure processing, foods containing high levels of solutes may need longer processing times or higher pressures to achieve inactivation of these fungi.     

Effect of high hydrostatic pressure on Listeria innocua 910 CECT inoculated into Ewe's milk.

Ewe's milk standardized to 6% fat was inoculated with Listeria innocua 910 CECT at a concentration of 10(7)CFU/ml and treated by high hydrostatic pressure. Treatments consisted of combinations of pressure (200, 300, 350, 400, 450, and 500 MPa), temperature (2, 10, 25, and 50 degrees C), and time (5, 10, and 15 min). To determine numbers of L. innocua, listeria selective agar base with listeria selective supplement and plate count agar was used. Low-temperature (2 degrees C) pressurizations produced higher L. innocua inactivation than treatments at room temperatures (25 degrees C). Pressures between 450 and 500 MPa for 10 to 15 min were needed to achieve reductions of 7 to 8 log units. The kinetics of destruction of L. innocua were first order with D-values of 3.12 min at 2 degrees C and 400 MPa and 4 min at 25 degrees C and 400 MPa. A baroprotective effect of ewe's milk (6% fat) on L. innocua was observed in comparison with other studies using different media and similar pressurization conditions.     

High sucrose concentration protects E. coli against high pressure inactivation but not against high pressure sensitization to the lactoperoxidase system

The inactivation of Escherichia coli by high hydrostatic pressure treatment at up to 550 MPa and 20 degrees C was studied in potassium phosphate buffer containing high concentrations of sucrose. E. coli strain MG1655 was pressure-sensitive in the absence of sucrose, but became highly pressure resistant in the presence of 10% to 50% (w/v) sucrose. The pressure resistance of E. coli strain LMM1010, a previously described derivative of MG1655 that is pressure resistant in the absence of sucrose, was further increased in the presence of sucrose, to a similar level as for strain MG1655 in the presence of sucrose. When cell suspensions of either strain were stored after pressure treatment for 24 h at 20 degrees C, a further reduction of the plate counts indicative of pressure induced sublethal injury was observed, that was positively correlated with pressure intensity and negatively with sucrose concentration. Addition of the lactoperoxidase system to the cell suspensions strongly enhanced high pressure inactivation of E. coli at high sucrose concentrations. Using a pressure intensity of only 250 MPa, both E. coli strains were sensitized for the lactoperoxidase system in up to 30% (w/v) sucrose, resulting in at least 10(6)-fold inactivation within 24 h or less after pressure treatment. For comparison, a pressure treatment at 250 MPa in the absence of the lactoperoxidase system did not cause any inactivation of either strain even in the absence of sucrose. At sucrose concentrations above 30% (w/v), no or very little inactivation occurred even in the presence of the lactoperoxidase system.     

Sucrose, sodium dodecyl sulfate, urea, and 2-mercaptoethanol affect the thermal inactivation of R-phycoerythrin.

Thermal inactivation kinetics (D- and z-values) of the algal protein, R-phycoerythrin (R-PE), were studied under different buffer conditions (pH 4.0, 7.0, and 10.0) and concentrations of sucrose, sodium dodecyl sulfate (SDS), urea, and 2-mercaptoethanol (ME). R-PE solutions were heated in capillary tubes at temperatures between 40 and 90 degrees C depending on buffer conditions. Thermal inactivation parameters for R-PE, calculated on the basis of fluorescence loss, were modified by addition of chemicals. Overall, sucrose and ME had a thermostabilizing effect, while SDS and urea decreased thermal stability of R-PE. The z-values ranged from 5.9 degrees C in 50 mM NaCl, 20 mM glycine buffer, pH 10.0, to 37.8 degrees C in 60% sucrose, 50 mM NaCl, 20 mM phosphate buffer, pH 7.0. The z-values obtained for R-PE closely matched the z-values of some target microorganisms in food processes, suggesting R-PE might be used as a time-temperature integrator to verify thermal processing adequacy.     

Modeling time to inactivation of Listeria monocytogenes in response to high pressure, sodium chloride, and sodium lactate

A mathematical model was developed to predict time to inactivation (TTI) by high pressure processing of Listeria monocytogenes in a broth system (pH 6.3) as a function of pressure (450 to 700 MPa), inoculum level (2 to 6 log CFU/ml), sodium chloride (1 or 2%), and sodium lactate (0 or 2.5%) from a 4°C initial temperature. Ten L. monocytogenes isolates from various sources, including processed meats, were evaluated for pressure resistance. The five most resistant strains were used as a cocktail to determine TTI and for model validation. Complete inactivation of L. monocytogenes in all treatments was demonstrated with an enrichment method. The TTI increased with increasing inoculum level and decreasing pressure magnitude, from 1.5 min at 700 MPa and 2 log CFU/ml, to 15 min at 450 MPa and 6 log CFU/ml. Neither NaCl nor sodium lactate significantly influenced TTI. The model was validated with ready-to-eat, uncured, Australian retail poultry products, and with product specially made at a U.S. Department of Agriculture, Food Safety and Inspection Service (FSIS)-inspected pilot plant in the United States. Data from the 210 individual product samples used for validation indicate that the model gives "fail-safe" predictions (58% with response as expected, 39% with no survivors where survivors expected, and only 3% with survivors where none were expected). This model can help manufacturers of refrigerated ready-to-eat meats establish effective processing criteria for the use of high pressure processing as a postlethality treatment for L. monocytogenes in accordance with FSIS regulations.     

High pressure assisted extraction of proteins from wet biomass of Arthrospira platensis (spirulina) – A kinetic approach

The application of high-pressure technology (100–600 MPa, 20 °C for up to 20 min) for cell disruption and consequent extraction of proteins -including C-phycocyanin- from the cyanobacteria Arthrospira platensis (spirulina), was investigated. Wet spirulina biomass was suspended in three different aqueous systems (deionized water, phosphate buffer, 10% sodium chloride solution). During a-24 h post processing storage period at 20 °C, the concentration of total soluble proteins and C-phycocyanin content and purity were measured. Color-spectrum and chroma analyses were also performed. The use of deionized water and phosphate buffer as processing/extraction media favoured the extraction process compared to the NaCl solution. Proteins extraction was significantly assisted by pressure. Equal/higher intensity than 400 MPa led to lower C-phycocyanin extraction yields, probably due to denaturation of proteins. High pressure conditions at 300 MPa for 10 min -using deionized water or phosphate buffer as medium- were selected as optimum, leading to higher extraction yields and purity extracts within ~2 h after processing.     

Quality characteristics of spent layer<Emphasis Type="Italic"> surimi</Emphasis> during frozen storage

The effects of different washing solutions and cryoprotectants on quality characteristics of spent layer surimi were studied during frozen storage at –18 °C. The highest (P<0.05) process yield was obtained with 0.5% sodium bicarbonate and 0.04 M sodium phosphate buffer solutions. While washing resulted in decreases (P<0.05) in protein, fat, cholesterol and ash contents, collagen and myofibrillar protein showed increases compared to the unwashed mince. A cryoprotectant mixture of 2% sucrose+2% sorbitol+0.3% sodium pyrophosphate resulted in higher water-holding capacity, except for surimi washed with 0.04 M sodium phosphate buffer solution, and lower cooking loss (P<0.05) than a cryoprotectant mixture of 4% sucrose+2.8% sorbitol. Increased lightness and decreased redness was observed as a result of washing with all solutions (P<0.05).     

Temperature-assisted pressure inactivation of Listeria monocytogenes in turkey breast meat

Ready-to-eat turkey breast meat samples were surface-inoculated with a five-strain cocktail of Listeria monocytogenes cultures to a final concentration of approximately 10(7) CFU/g. The inoculated meat samples were vacuum-packaged and pressure treated at 300 MPa for 2 min, 400 MPa for 1 min, and 500 MPa for 1 min at initial sample temperatures of 1, 10, 20, 30, 40, 50, and 55 degrees C. L. monocytogenes was most resistant to pressure at temperatures between 10 and 30 degrees C. As temperature decreased below 10 degrees C or increased over 30 degrees C, its pressure sensitivity increased. This enhanced inactivation effect was more pronounced when meat samples were treated at higher temperature than at lower temperature. For example, a 1-min treatment of 500 MPa at 40 degrees C reduced the counts by 3.8 log(10), while at 1 and 20 degrees C the same treatment reduced counts by 1.4 and 0.9 log(10), respectively (P<0.05). The survival curves of L. monocytogenes were obtained at 300 MPa and 55 degrees C, 400 MPa and 50 degrees C, and 500 MPa and 40 degrees C. With increasing treatment time, the three survival curves showed a rapid initial drop in bacteria counts with a diminishing inactivation rate or tailing effect. The survival data were fitted with a linear and a nonlinear, Weibull, models. The Weibull model consistently produced better fit to the survival data than the linear model.     

A predictive model for the influence of food components on survival of Listeria monocytogenes LM 54004 under high hydrostatic pressure and mild heat conditions

The combination of high hydrostatic pressure with mild temperature was explored to achieve a predictive model of microbial inactivation in food matrix processing. The pressure processing conditions were fixed at 448 MPa for 11 min at the treatment temperature of 41 degrees C, which have been determined as the optimum processing conditions considering six log-cycle reductions of Listeria monocytogenes. Based on the results, response surface methodology (RSM) was performed in the present work, the influence of food components like soybean protein (0-5.00%), sucrose (0.25-13.25%), bean oil (0-10.00%), and pH (4-10) of the food matrix on survival of L. monocytogenes by high pressure and mild heat was studied, and a quadratic predictive model for the influence of food components and pH of food matrix on L. monocytogenes reduction by high pressure and mild heat was built with RSM accurately. The experimental results showed that the efficiency of L. monocytogenes reduction in milk buffer and food matrix designed in the present work, under the HPP treatment process parameters described above, were different. The soybean protein (P=0.0086), sucrose (P<0.0001), and pH (P=0.0136) significantly affected reduction of L. monocytogenes, but the effect of bean oil on reduction of L. monocytogenes was not significant (P=0.1028). The predictive model is significant since the level of significance was P<0.0001 and the calculated F value (11.53) is much greater than the tabulated F value (F(0.01 (14, 5))=9.77). Moreover, the adequacy of the predictive model equation for predicting the level of L. monocytogenes reduction was verified effectively by the validation data.     

Pressure death and tailing behavior of Listeria monocytogenes strains having different barotolerances

The objectives of this study were to investigate the variability among Listeria monocytogenes strains in response to high-pressure processing, identify the most resistant strain as a potential target of pressure processing, and compare the inactivation kinetics of pressure-resistant and pressure-sensitive strains under a wide range (350 to 800 MPa) of pressure treatments. The pressure resistance of Listeria innocua and nine strains of L. monocytogenes was compared at 400 or 500 MPa and 30 degrees C. Significant variability among strains was observed. The decrease in log CFU/ml during the pressure treatment was from 1.4 to 4.3 at 400 MPa and from 3.9 to >8 at 500 MPa. L. monocytogenes OSY-8578 exhibited the greatest pressure resistance, Scott A showed the greatest pressure sensitivity, and L. innocua had intermediate resistance. On the basis of these findings, L. monocytogenes OSY-8578 is a potential target strain for high-pressure processing efficacy studies. The death kinetics of L. monocytogenes Scott A and OSY-8578 were investigated at 350 and 800 MPa. Survivors at 350 MPa were enumerated by direct plating, and survivors at 800 MPa were enumerated by the most-probable-number technique. Both pressure-resistant and pressure-sensitive strains exhibited non-first-order death behavior, and excessive pressure treatment did not eliminate the tailing phenomenon.     

Model System Studies on N-Nitrosamine Formation in Cured Meats: The Effect of Curing Solution Ingredients

Solutes commonly employed in curing solutions were evaluated for their effect on nitrosamine formation utilizing a ground pork model system. Sodium erythorbate and sodium ascorbate were found to be equally effective in reducing nitrosopyrrolidine levels. The incorporation of sodium tripolyphosphate or sucrose had no detectable effect on nitrosamine formation. However, sodium chloride, the solute present at the highest concentration in bacon curing solutions, exhibited a concentration dependent inhibition of nitrosopyrrolidine formation. Nitrosopyrrolidine levels in samples containing no added sodium chloride were found to be 50% higher than samples processed with 1.5% added salt.     

High pressure inactivation of microorganisms inoculated into ovine milk of different fat contents

High hydrostatic pressure inactivation of Escherichia coli, Pseudomonas fluorescens, Listeria innocua, Staphylococcus aureus, and Lactobacillus helveticus were studied. These microorganisms were inoculated at a concentration between 10(7) and 10(8) cfu/ml in Ringer solution and in ovine milk adjusted to 0, 6, and 50% fat content to evaluate the baroprotective effect of fat content on inactivation of microorganisms. Treatments of pressurization consisted of combinations of pressure (100 to 500 MPa) and temperature (4, 25, and 50 degrees C) for 15 min. Gram-negative microorganisms were more sensitive than were Gram-positive ones (more destruction P. fluorescens > E. coli > or = List. innocua > Lb. helveticus > S. aureus). Pressurizations at low temperature (4 degrees C) produced greater inactivation on P. fluorescens, List. innocua, and Lb. helveticus than at room temperature (25 degrees C), whereas for E. coli and S. aureus the results were opposite. Ovine milk per se (0% fat) showed a baroprotective effect on all microorganisms, but percentage of fat (6 and 50%) did not show a progressive baroprotective effect in all pressurization conditions or for all microorganisms.     

Evaluation of chemical and physical (high-pressure and temperature) treatments to improve the safety of minimally processed mung bean sprouts during refrigerated storage

The objective of this study was to compare the effects of combined high hydrostatic pressure and temperature treatments with different chemical sanitation treatments (water, sodium hypochlorite, and hydrogen peroxide) on the microbiological properties of mung bean sprouts. In a first study, the raw product was subjected to several combined high-pressure and temperature treatments for calculating a mathematical model by a response surface methodology. The number of pressure-temperature (150 to 400 MPa; 20 to 40 degrees C) combinations was limited to 10. In addition, a model system consisting of mung bean sprout juice was inoculated with Listeria monocytogenes (CECT 4032). Microbial inactivation with this model system was also investigated by a response surface methodology. The highest aerobic mesophilic bacteria and L. monocytogenes inactivation was achieved at maximum pressure and temperature (5.5 and 1.8 log cycles, respectively). In a second study, the effect of five different processing lines on the microbial load reduction of minimally processed mung bean sprouts during refrigerated storage was studied. All treatments reduced the initial population of aerobic mesophilic bacteria and fecal coliforms, with the physical treatment of 400 MPa and 40 degrees C being the most effective, showing initial reductions of 5.8 and 7.8 log CFU/ g, respectively. Recovery of bacteria from sprouts treated under these conditions was not observed during storage. However, the sprouts that received washing treatments with water, sodium hypochlorite, and hydrogen peroxide exhibited increases in aerobic mesophilic and fecal coliform counts after 3 days of storage at 4 degrees C.     

Pressure inactivation kinetics of Yersinia enterocolitica ATCC 35669

The survival curves of Yersinia enterocolitica ATCC 35669 inactivated by high hydrostatic pressure were obtained at four pressure levels (300, 350, 400, and 450 MPa) in sodium phosphate buffer (0.1 M, pH 7.0) and four pressure levels (350, 400, 450, 500 MPa) in UHT whole milk. Tailing was observed in all the survival curves. A linear model and three nonlinear models were fitted to these data and the performances of these models were compared. The linear regression model for survival curves at four pressure levels had regression coefficients (R2) values of 0.785-0.962 and mean square error (MSE) of 0.265-0.893. A residual plot strongly suggested that a linear regression function was not appropriate as there was strong curvature in the plotted data. The nonlinear regression model using the log-logistic had R2 values of 0.946-0.982 and MSE values of 0.110-0.320. The Weibull model had R2 values of 0.944-0.975 and MSE values of 0.153-0.349. These results indicated that both were better models to describe the pressure inactivation kinetics of Y. enterocolitica in milk and buffer. Among the three nonlinear models studied, the modified Gompertz model produced the poorest fit to data. The number of parameters of the log-logistic model was reduced from four to two so that the model was greatly simplified. The reduced log-logistic model still produced a fit comparable to the full model. Since pressure had no significant effect on the shape factors of the Weibull model at the pressure levels of 300-400 MPa for buffer and 400-500 MPa for milk, models were developed to predict survival curves of Y. enterocolitica at pressures different from the experimental pressures.