Investigating the Effect of Rosemary Essential Oil, Supercritical CO2 Processing and Their Synergism on the Quality and Microbial Inactivation of Chicken Breast Meat.

Fresh chicken meat is a very perishable good, even at refrigerated storage conditions, due to psychrophilic microbial growth and physicochemical changes. The present study focuses on the use of rosemary (Rosmarinus officinalis L.) essential oil (REO), supercritical CO2 processing and their synergism to increase the microbial inactivation in chicken breast meat. E. coli and L. innocua were inoculated on the chicken breast surface, and the inactivation effects of two different processes, namely SC-CO2 and SC-MAPCO2, were compared with or without the addition of REO. Moreover, the impact of the treatments on the superficial color of the meat was considered. The study demonstrated a synergic effect with 1% REO and supercritical CO2 for the inactivation of E. coli on chicken meat, while for L. innocua, there was no synergism. Regarding SC-CO2 treatment, the E. coli reduction was 1.29 and 3.31 log CFU/g, while for L. innocua, it was 1.42 and 1.11 log CFU/g, respectively, without and with the addition of 1.0% of REO. The same amount of REO allowed us to obtain a reduction of 1.3 log CFU/g of E. coli when coupled with SC-MAPCO2. For L. innocua, no reduction was obtained, either with SC-MAPCO2 or together with REO. The synergism of SC-MAPCO2 with 1% REO was confirmed for the total psychrophilic bacteria, demonstrating a strong dependence on the microorganism. The color modification induced by the SC-MAPCO2 process was lower than the SC-CO2 treatment. Overall, this study demonstrated a possible synergism of the technologies which can support the development of innovative methods to improve the safety and shelf-life of chicken breast meat.

The effect of supercritical carbon dioxide on the physiochemistry, endogenous enzymes, and nutritional composition of fruit and vegetables and its prospects for industrial application: a overview.

Consumers have an increasing demand for fruit and vegetables with high nutritional value worldwide. However, most fruit and vegetables are vulnerable to quality loss and spoilage during processing, transportation, and storage. Among the recently introduced emerging technologies, supercritical carbon dioxide (SCCO2) has been extensively utilized to treat and maintain fruit and vegetables mainly due to its nontoxicity, safety, and environmentally friendly. SCCO2 technology generates low processing costs and mild processing conditions (temperature and pressure) that allow for the application of CO2 at a supercritical state. This review aimed to summarize the current knowledge on the influence of SCCO2 technology on the quality attributes of fruit and vegetable products, such as physicochemical properties (pH, color, cloud, particle size distribution, texture), sensory quality, and nutritional composition (ascorbic acid, phenolic compounds, anthocyanins, carotenoids, and betalains). In addition, the effects and mechanisms of the SCCO2 technique on endogenous enzyme inactivation (polyphenol oxidase, peroxidase, and pectin methylesterase) were also elucidated. Finally, the prospects of the SCCO2 technique for industrial application was discussed from the economic and regulatory aspect.

Training in tools to develop quantitative microbial risk assessment along the food chain of Spanish products.

Food safety is a widespread challenge. Every year it is estimated that almost 1 in 10 people in the world fall ill after eating contaminated food resulting in over 400,000 deaths. The risk of outbreaks is higher when consuming ready-to-eat (RTE) products because they are eaten without a further cooking process that could inactivate pathogenic microorganisms. Hence, food processing is essential to increase the safety of RTE products. Microbiological risk assessment (MRA) integrates food science, microbiology and data science to provide a comprehensive understanding of the safety of the food system. MRA provides qualitative and/or quantitative information to decision makers, which might promote the adoption of better food practices. In this contest, this project aims to study and implement tools for quantitative microbial risk assessment (QMRA) of food products along the food chain. A common RTE product (cured ham) from Spain was used as a case study. Following, the exposure assessment model was implemented using mathematical models and statistical software to describe the microbial behaviour along the food chain. The study presents the possibility to identify the risk exposure in different scenarios (e.g. growth during different storage conditions, inactivation induced by traditional or innovative decontamination techniques), showing the flexibility of the predictive tools developed.

Promoting the preservation of strawberry by supercritical CO2 drying.

This work aimed to investigate the supercritical CO2 (ScCO2) drying of strawberries and its effect on enzymatic, chemical and microbial stability. Process conditions influenced the final weight loss (WL), water activity (aw) and the inactivation of polyphenol oxidase (PPO) and peroxidase (POD). At 40 °C, an efficient drying (WL > 92 %, aw < 0.34) and a complete enzymatic (POD and PPO activity) inactivation can be achieved using several combinations of pressure, time and flow rate. ScCO2 dried strawberry at 40 °C, 13.3 MPa, 7 h and 19 kg/h flow rate maintain the total content of Vitamin C (358.5 mg/100 g), 95 % of total anthocyanin (61.68 mg/100 g) and 76 % of total flavonoids (25.85 mg/100 g) in comparison with fresh samples. Foodborne pathogens (E.coli O157:H7, Salmonella enterica and Listeria monocytogenes) inoculated at high concentration (≥6 log CFU/g) were undetected after the process. Overall results are promising for the development of a novel low temperature drying process for the production of healthy and safe snack.

Increasing the Safety and Storage of Pre-Packed Fresh-Cut Fruits and Vegetables by Supercritical CO2 Process.

This work presents a feasibility lab-scale study for a new preservation method to inactivate microorganisms and increase the shelf life of pre-packed fresh-cut products. Experiments were conducted on coriander leaves and fresh-cut carrots and coconut. The technology used the combination of hydrostatic pressure (<15 MPa), low temperature (≤45 °C), and CO2 modified atmosphere packaging (MAP). The inactivation was achieved for the naturally present microorganisms (total mesophilic bacteria, yeasts and molds, total coliforms) and inoculated E. coli. Yeasts and molds and coliform were under the detection limit in all the treated samples, while mesophiles were strongly reduced, but below the detection limit only in carrots. Inoculated E. coli strains were completely inactivated (>6.0 log CFU/g) on coconut, while a reduction >4.0 log CFU/g was achieved for carrots and coriander. For all the treated products, the texture was similar to the fresh ones, while a small alteration of color was detected. Microbiological stability was achieved for up to 14 days for both fresh-cut carrots and coconut. Overall, the results are promising for the development of a new mild and innovative food preservation technique for fresh food.

Optimization of the Appearance Quality in CO2 Processed Ready-to-Eat Carrots through Image Analysis.

A high-pressure CO2 process applied to ready-to-eat food products guarantees an increase of both their microbial safety and shelf-life. However, the treatment often produces unwanted changes in the visual appearance of products depending on the adopted process conditions. Accordingly, the alteration of the visual appearance influences consumers' perception and acceptability. This study aims at identifying the optimal treatment conditions in terms of visual appearance by using an artificial vision system. The developed methodology was applied to fresh-cut carrots (Daucus carota) as the test product. The results showed that carrots packaged in 100% CO2 and subsequently treated at 6 MPa and 40 °C for 15 min maintained an appearance similar to the fresh product for up to 7 days of storage at 4 °C. Mild appearance changes were identified at 7 and 14 days of storage in the processed products. Microbiological analysis performed on the optimal treatment condition showed the microbiological stability of the samples up to 14 days of storage at 4 °C. The artificial vision system, successfully applied to the CO2 pasteurization process, can easily be applied to any food process involving changes in the appearance of any food product.

Effect of CO2 Preservation Treatments on the Sensory Quality of Pomegranate Juice.

Due to the interest in identifying cost-effective techniques that can guarantee the microbiological, nutritional, and sensorial aspects of food products, this study investigates the effect of CO2 preservation treatment on the sensory quality of pomegranate juice at t0 and after a conservation period of four weeks at 4 °C (t28). The same initial batch of freshly squeezed non-treated (NT) juice was subjected to non-thermal preservation treatments with supercritical carbon dioxide (CO2), and with a combination of supercritical carbon dioxide and ultrasound (CO2-US). As control samples, two other juices were produced from the same NT batch: A juice stabilized with high pressure treatment (HPP) and a juice pasteurized at high temperature (HT), which represent an already established non-thermal preservation technique and the conventional thermal treatment. Projective mapping and check-all-that-apply methodologies were performed to determine the sensory qualitative differences between the juices. The volatile profile of the juices was characterized by gas chromatography-mass spectrometry. The results showed that juices treated with supercritical CO2 could be differentiated from NT, mainly by the perceived odor and volatile compound concentration, with a depletion of alcohols, esters, ketones, and terpenes and an increase in aldehydes. For example, in relation to the NT juice, limonene decreased by 95% and 90%, 1-hexanol decreased by 9% and 17%, and camphene decreased by 94% and 85% in the CO2 and CO2-US treated juices, respectively. Regarding perceived flavor, the CO2-treated juice was not clearly differentiated from NT. Changes in the volatile profile induced by storage at 4 °C led to perceivable differences in the odor quality of all juices, especially the juice treated with CO2-US, which underwent a significant depletion of all major volatile compounds during storage. The results suggest that the supercritical CO2 process conditions need to be optimized to minimize impacts on sensory quality and the volatile profile.

Research Note: Microbial inactivation of raw chicken meat by supercritical carbon dioxide treatment alone and in combination with fresh culinary herbs.

The objective of the present study was to assess the potential synergistic effect between supercritical carbon dioxide (SC-CO2) and fresh culinary herbs (Coriandrum sativum and Rosmarinus officinalis) on the microbial inactivation of raw chicken meat. The microbiological inactivation was performed on Escherichia coli and natural flora (total mesophilic bacteria, yeasts, and molds). High pressure treatments were carried out at 40°C, 80 or 140 bar from 15 to 45 min. Microbial inactivation had a strong dependence on treatment time, achieving 1.4 log CFU/g reduction of E. coli after 15 min, and up to 5 log after 45 min, while a pressure increase from 80 up to 140 bar was not significant on the microbial inactivation. Mesophilic microorganisms were strongly reduced (>2.6 log CFU/g) after 45 min, and yeasts and molds were below the detection limits of the technique (<100 CFU/g) in most cases. The combination of fresh herbs together with SC-CO2 treatment did not significantly increase the inactivation of either E. coli or natural flora, which was similar to the SC-CO2 alone. The synergistic effect was obtained on the inactivation of E. coli using a proper concentration of coriander essential oil (EO) (0.5% v/w), while rosemary EO did not show a significant effect. Color analysis after the treatment showed an increment of lightness (L*), and a decrease of redness (a*) on the surface of the sample, making the product visually similar to cooked meat. Texture analysis demonstrated the modification of the texture parameters as a function of the process pressure making the meat more similar to the cooked one.

Enzymatic, physicochemical, nutritional and phytochemical profile changes of apple (Golden Delicious L.) juice under supercritical carbon dioxide and long-term cold storage.

The impact of supercritical carbon dioxide (SCCD) (10-60 MPa/45 °C/30 min) and subsequent 10 weeks storage at 4 °C on polyphenol oxidase (PPO), peroxidase (POD) activities, phenolic profile, vitamin C, sugars, physicochemical properties of cloudy apple juices was investigated. No significant changes in sugars and total polyphenols were observed, whereas significant degradation (≈28%) of vitamin C and individual polyphenols (≈18%) was noted after SCCD treatment. After 4 weeks storage only 34% of vitamin C was retained and no vitamin C was detected after this time. Ten weeks of storage caused hydrolysis of sucrose in 15%, whereas degradation of individual polyphenols ranged from 43 to 50% depending on the pressure applied. The highest pressure was applied the highest retention of polyphenols was observed. The lightness of juice significantly increased by 15% after SCCD and decreased during storage. Moreover, the synergistic effect of both enzymes with chlorogenic acid and catechol was found.

In Situ Raman Analysis of CO2-Assisted Drying of Fruit-Slices.

This work explores the feasibility of applying in situ Raman spectroscopy for the online monitoring of the supercritical carbon dioxide (SC-CO2) drying of fruits. Specifically, we investigate two types of fruits: mango and persimmon. The drying experiments were carried out inside an optical accessible vessel at 10 MPa and 313 K. The Raman spectra reveal: (i) the reduction of the water from the fruit slice and (ii) the change of the fruit matrix structure during the drying process. Two different Raman excitation wavelengths were compared: 532 nm and 785 nm. With respect to the quality of the obtained spectra, the 532 nm excitation wavelength was superior due to a higher signal-to-noise ratio and due to a resonant excitation scheme of the carotenoid molecules. It was found that the absorption of CO2 into the fruit matrix enhances the extraction of water, which was expressed by the obtained drying kinetic curve.

Supercritical CO2 induces marked changes in membrane phospholipids composition in Escherichia coli K12.

Supercritical carbon dioxide (SC-CO2) treatment is one of the most promising alternative techniques for pasteurization of both liquid and solid food products. The inhibitory effect of SC-CO2 on bacterial growth has been investigated in different species, but the precise mechanism of action remains unknown. Membrane permeabilization has been proposed to be the first event in SC-CO2-mediated inactivation. Flow cytometry, high performance liquid chromatography­electrospray ionization­mass spectrometry and NMR analyses were performed to investigate the effect of SC-CO2 treatment on membrane lipid profile and membrane permeability in Escherichia coli K12. After 15 min of SC-CO2 treatment at 120 bar and 35 °C, the majority of bacterial cells dissipated their membrane potential (95 %) and lost membrane integrity, as 81 % become partially permeabilized and 18 % fully permeabilized. Membrane permeabilization was associated with a 20 % decrease in bacterial biovolume and to a strong (>50 %) reduction in phosphatidylglycerol (PG) membrane lipids, without altering the fatty acid composition and the degree of unsaturation of acyl chains. PGs are thought to play an important role in membrane stability, by reducing motion of phosphatidylethanolamine (PE) along the membrane bilayer, therefore promoting the formation of inter-lipid hydrogen bonds. In addition, the decrease in intracellular pH induced by SC-CO2 likely alters the chemical properties of phospholipids and the PE/PG ratio. Biophysical effects of SC-CO2 thus cause a strong perturbation of membrane architecture in E. coli, and such alterations are likely associated with its strong inactivation effect.

Comparison of quantitative PCR and flow cytometry as cellular viability methods to study bacterial membrane permeabilization following supercritical CO2 treatment.

Foodborne illness due to bacterial pathogens is increasing worldwide as a consequence of the higher consumption of fresh and minimally processed food products, which are more easily cross-contaminated. The efficiency of food pasteurization methods is usually measured by c.f.u. plate counts, a method discriminating viable from dead cells on the basis of the ability of cells to replicate and form colonies on standard growth media, thus ignoring viable but not cultivable cells. Supercritical CO2 (SC-CO2) has recently emerged as one of the most promising fresh food pasteurization techniques, as an alternative to traditional, heat-based methods. In the present work, using three SC-CO2-treated foodborne bacteria (Listeria monocytogenes, Salmonella enterica and Escherichia coli) we tested and compared the performance of alternative viability test methods based on membrane permeability: propidium monoazide quantitative PCR (PMA-qPCR) and flow cytometry (FCM). Results were compared based on plate counts and fluorescent microscopy measurements, which showed that the former dramatically reduced the number of cultivable cells by more than 5 log units. Conversely, FCM provided a much more detailed picture of the process, as it directly quantifies the number of total cells and distinguishes among three categories, including intact, partially permeabilized and permeabilized cells. A comparison of both PMA-qPCR and FCM with plate count data indicated that only a fraction of intact cells maintained the ability to replicate in vitro. Following SC-CO2 treatment, FCM analysis revealed a markedly higher level of bacterial membrane permeabilization of L. monocytogenes with respect to E. coli and S. enterica. Furthermore, an intermediate permeabilization state in which the cellular surface was altered and biovolume increased up to 1.5-fold was observed in L. monocytogenes, but not in E. coli or S. enterica. FCM thus compared favourably with other methods and should be considered as an accurate analytical tool for applications in which monitoring bacterial viability status is of importance, such as microbiological risk assessment in the food chain or in the environment.

Optimization of supercritical carbon dioxide treatment for the inactivation of the natural microbial flora in cubed cooked ham.

This study aims to investigate the effects of supercritical carbon dioxide (SC-CO2) treatment on the inactivation of the natural microbial flora in cubed cooked ham. Response surface methodology with a central composite design was applied to determine the optimal process conditions and investigate the effect of three independent variables (pressure, temperature and treatment time). Additionally, analyses of texture, pH and color together with a storage study of the product were performed to determine its microbial and qualitative stability. Response surface analysis revealed that 12 MPa, 50 °C, 5 min were the optimal conditions to obtain about 3.0, 1.6, and 2.5 Log(CFU/g) reductions of mesophilic aerobic bacteria, psychrophilic bacteria and lactic acid bacteria respectively. Inactivation to undetectable levels of yeasts and molds and coliforms was also obtained. A storage study of 30 days at 4 °C was carried out on the treated product (12 MPa, 50 °C, 5 min) monitoring microbial growth, pH, texture, and color parameters (L*, a*, b* and ΔE). Microbial loads slightly increased and after 30 days of storage reached the same levels detected in the fresh product. Color parameters (L*, a*, b*) showed slight variations while pH and texture did not change significantly. On the basis of the results obtained, SC-CO2 can be considered a promising technique to microbiologically stabilize cubed cooked ham and, in general, cut/sliced meat products without affecting its quality attributes.

Effect of supercritical carbon dioxide pasteurization on natural microbiota, texture, and microstructure of fresh-cut coconut.

The objective of the present study was the evaluation of the effectiveness of supercritical carbon dioxide (SC-CO(2)) as a nonthermal technology for the pasteurization of fresh-cut coconut, as an example of ready-to-eat and minimally processed food. First, the inactivation kinetics of microbiota on coconut were determined using SC-CO(2) treatments (pressures at 8 and 12 MPa, temperatures from 24 to 45 °C, treatment times from 5 to 60 min). Second, the effects of SC-CO(2) on the hardness and microstructure of fresh-cut coconut processed at the optimal conditions for microbial reduction were investigated. SC-CO(2) treatment of 15 min at 45 °C and 12 MPa induced 4 log CFU/g reductions of mesophilic microorganisms, lactic acid bacteria, total coliforms, and yeasts and molds. The hardness of coconut was not affected by the treatment but the samples developed an irregular and disorderly microstructure. Results suggested the potential of SC-CO(2) in preserving fresh-cut fruits and ready-to-eat products.

Carbon dioxide induced silk protein gelation for biomedical applications.

We present a novel method to fabricate silk fibroin hydrogels using high pressure carbon dioxide (CO(2)) as a volatile acid without the need for chemical cross-linking agents or surfactants. The simple and efficient recovery of CO(2) post processing results in a remarkably clean production method offering tremendous benefit toward materials processing for biomedical applications. Further, with this novel technique we reveal that silk protein gelation can be considerably expedited under high pressure CO(2) with the formation of extensive ß-sheet structures and stable hydrogels at processing times less than 2 h. We report a significant influence of the high pressure CO(2) processing environment on silk hydrogel physical properties such as porosity, sample homogeneity, swelling behavior and compressive properties. Microstructural analysis revealed improved porosity and homogeneous composition among high pressure CO(2) specimens in comparison to the less porous and heterogeneous structures of the citric acid control gels. The swelling ratios of silk hydrogels prepared under high pressure CO(2) were significantly reduced compared to the citric acid control gels, which we attribute to enhanced physical cross-linking. Mechanical properties were found to increase significantly for the silk hydrogels prepared under high pressure CO(2), with a 2- and 3-fold increase in the compressive modulus of the 2 and 4 wt % silk hydrogels over the control gels, respectively. We adopted a semiempirical theoretical model to elucidate the mechanism of silk protein gelation demonstrated here. Mechanistically, the rate of silk protein gelation is believed to be a function of the kinetics of solution acidification from absorbed CO(2) and potentially accelerated by high pressure effects. The attractive features of the method described here include the acceleration of stable silk hydrogel formation, free of residual mineral acids or chemical cross-linkers, reducing processing complexity, and avoiding adverse biological responses, while providing direct manipulation of hydrogel physical properties for tailoring toward specific biomedical applications.

Terminal sterilization of BisGMA-TEGDMA thermoset materials and their bioactive surfaces by supercritical CO2.

The development of biomaterials endowed with bioactive features relies on a simultaneous insight into a proper terminal sterilization process. FDA recommendations on sterility of biomaterials are very strict: a sterility assurance level (SAL) of 10(-6) must be guaranteed for biomaterials to be used in human implants. In the present work, we have explored the potential of supercritical CO(2) (scCO(2)) in the presence of H(2)O(2) as a low-temperature sterilization process for thermoset materials and their bioactive surfaces. Different conditions allowing for terminal sterilization have been screened and a treatment time-amount of H(2)O(2) relationship proposed. The selected terminal sterilization conditions did not notably modify the mechanical properties of the thermoset nor of their fiber-reinforced composites. This was confirmed by µCT analyses performed prior to and after the treatment. On the contrary, terminal sterilization in the presence of H(2)O(2) induced a slight decrease in the surface hardness. The treatment of the thermoset material with scCO(2) led to a reduction in the residual unreacted monomers content, as determined by means of high performance liquid chromatography (HPLC) analyses. Finally, it was found that a thermoset coated with a polysaccharide layer containing silver nanoparticles maintained a very high antimicrobial efficacy even after the scCO(2)-based terminal sterilization.

Porous poly(D,L-lactic acid) foams with tunable structure and mechanical anisotropy prepared by supercritical carbon dioxide.

The design and tunability of tissue scaffolds, such as pore size and geometry, is crucial to the success of an engineered tissue replacement. Moreover, the mechanical nature of a tissue scaffold should display properties similar to the tissue of interest; therefore, tunability of the foam mechanical properties is desirable. Polymeric foams prepared using supercritical carbon dioxide as a blowing agent has emerged in recent years as a promising technique to prepare porous scaffolds. While a number of groups have reported on the tailoring of scaffold morphologies by using gas foaming techniques, few have considered the effects of such processing conditions on the physical and mechanical anisotropy achieved. The aim of this study was to demonstrate the tunability of the structure and mechanical anisotropy of foams prepared using a variety of different gas foaming conditions. Porous poly(D,L lactic acid) foams were prepared by the systematic adjustment of processing conditions, namely pressure, temperature and venting time, resulting in an extensive range of scaffold morphologies. Characterization of sample anisotropy was achieved by mechanical evaluation of foam specimens both longitudinal and transverse to the foaming direction. The obtained mechanical properties demonstrated a strong dependence of the processing conditions on mechanical anisotropy and performance. Furthermore, results indicate that factors other than pore geometry may be necessary to define the mechanical behavior of the foam specimens. The favorable compressive moduli, coupled with large degrees of anisotropy, suggests these foams may have suitable application as scaffolds for bone tissue engineering.

High pressure gases: role of dynamic intracellular pH in pasteurization.

Certain dense gases, including CO(2) and N(2)O, are known to deactivate food pathogens safely. Complete deactivation requires disabling intracellular metabolic pathways by extracellular processing that causes membrane disruption, irreversible denaturation of proteins, or extraction of cell contents. At present, neither the precise physical and metabolic mechanisms nor their kinetics behind dense gas pasteurization are known. The mechanisms depend strongly on both the organism and the environment and may combine membrane disruption, membrane permeabilization, and altering pH. Herein we elucidate the mechanisms of dense gas inactivation with the aid of a novel approach for measuring intracellular pH (pH(i)) under high gas pressure. Using a pH-sensitive GFP-variant of S. cerevisiae as the probe, we demonstrate that membrane permeabilization by a non-acidic gas, N(2)O, contributes to inactivation but at a rate that is relatively low compared to CO(2). CO(2) not only permeabilizes the membrane but also brings about a rapid drop in pH(i), leading to greater deactivation. Mechanistic understanding is vital to develop safe and effective dense gas technologies for food treatment. Knowledge of pH(i) is also important in other cellular processes, including enzyme activity, gene transcription, and protein synthesis. The GFP technique has been demonstrated to be versatile even under pressure.

Mathematical modeling of yeast inactivation of freshly squeezed apple juice under high-pressure carbon dioxide.

The number of data sets available in literature regarding inactivation kinetic of microorganisms at low temperature, demonstrate an increasing attention to new technologies for food preservation at ambient temperature. Nevertheless, no reliable modeling, capable to describe complex inactivation curves, such as the ones due to dense gas pasteurization with a log-linear behavior, have been developed thus far. In this respect, the main aim of this work is to analyze and model experimental data of dense carbon dioxide yeast pasteurization of natural apple juice at different condition of temperature (25-36°C) and sample volume (5-10 ml). The Weibull model modified by Albert and Mafart was verified to be an interesting model capable to take into account CO2 inactivation kinetic, with a first phase with a shoulder, a second phase with a log-linear shape, and a final phase with a tailing with either a non-zero or a zero asymptote. Clearly, the results obtained shows that an increase in temperature decreases the time needed for the same inactivation efficiency; the residual yeast concentration of N(RES), a thermodynamic parameter, results volume independent, and temperature dependent; the treatment time required to reach 90% of inactivation results temperature dependent, with a sample volume of 5 ml; at 100 bar - 25°C-10 mL a shoulder effect is evident in the inactivation kinetic. The model can be considered a new useful tool to predict new CO2 pasteurization data at different operative conditions.

Determination of extracellular and intracellular pH of Bacillus subtilis suspension under CO2 treatment.

In this study, we consider the effect of carbon dioxide (CO(2)) on the intracellular and extracellular pH of a saline solution of a test-microorganisms Bacillus subtilis. The cytoplasmatic pH was determined by means of a flow cytometry with the fluorescent probe 5(and 6-)-carboxyfluorescein ester (cFSE). The physiological suspension of cells with the addition of the probe was first exposed to high pressure CO(2) for 5 min at different temperatures. The flow cytometry analysis indicated an intracellular depletion inside the cell caused by the action of CO(2), down to 3, the depletion being dependent on inactivation ratio. In addition, the extracellular pH was determined theoretically by means of the statistical associated fluid theory equation of state (SAFT EOS): it was demonstrated that CO(2) under pressure dissolves into liquid phase and acidifies the medium down to 3 at 80 bar and 303.15K. The results show a strong influence between extracellular and intracellular pH, and lead to the conclusion that a strong reduction of the pH homeostasis of the cell can be claimed as one of the most probable cause of inactivation of CO(2) pasteurization.