Pressure inactivation of fungal spores in aqueous model solutions and in real food systems

Abstract

Conidiospores from Penicillium expansum and ascospores from Eurotium repens were exposed to high hydrostatic pressure in isotonic salt solution, apple and broccoli juice. Kinetic measurements were done at 4,25 and 40 or 45°C. The shape of the inactivation curves was strongly dependent on the temperature. Asco- and conidiospores were found to behave in a contrary way. The fastest reduction for conidiospores was found at 4°C, for ascospores best inactivation was achieved at 45°C. High pressure inactivation of spores in apple or broccoli juice was nearly the same as in isotonic salt solution.     

Moderate hydrostatic pressure–temperature combinations for inactivation of Bacillus subtilis spores

We report the effect of using moderate hydrostatic pressure, 40–140?MPa, at moderate temperature (38–58°C) to inactivate Bacillus subtilis spores in McIlvaine's citric phosphate buffer at pH 6. We have investigated several parameters: pressure applied, holding time, pressure cycling, and temperature. The kinetics of spore inactivation is reported. The results show that spore inactivation is exponentially proportional to the time the sample is exposed to pressure. Spore germination and inactivation occur at the hydrostatic pressures/temperature combinations we explored. Cycling the pressure while keeping the total time at high pressure constant does not significantly increase spore inactivation. We show that temperature increases spore inactivation at two different rates; a slow rate below 33°C, and at a more rapid rate at higher temperatures. Increasing pressure leads to an increase in spore inactivation below 95?MPa; however, further increases in pressure give a similar rate kill. The time dependence of the effect of pressure is consistent with the first-order model (R²?>?0.9). The thermal resistance values (Z_T) of B. subtilis spores are 30°C, 37°C, and 40°C at 60, 80, 100?MPa. The increase in Z_T value at higher pressures indicates lower temperature sensitivity. The pressure resistance values (Z_P) are 125, 125 and 143?MPa at 38°C, 48°C, and 58°C. These Z_P values are lower than those reported for B. subtilis spores in the literature, which indicates higher sensitivity at pressures less than about 140?MPa. We show that at temperatures <60°C, B. subtilis spores are inactivated at pressures below 100?MPa. This finding could have implications for the design of the sterilization equipment.     

Evaluation of the importance of germinative cycles for destruction of bacillus cereus spores in miniature cheeses

Our objective was to determine the effect of high pressure on inactivation of spores of Bacillus cereus ATCC 9139 inoculated into cheese made of raw cow's milk. Inoculated miniature cheeses were manufactured under controlled bacteriological conditions, vacuum packed and kept at 8 °C for 15 days after pressure treatment. Cheeses were submitted to pressures of 300, 400 or 500 MPa at 30 °C, during 15 min. Some of them were treated with a germination cycle of 60 MPa at 30 °C for 210 min. Lethality was calculated comparing surviving sample counts to control ones. Adding the germinative cycle resulted in higher efficiency, and when applied with 500 MPa, lethality reached 2.0 log cfu/mL. We saw that with both cycles initial counts of spores diminish, but all of them were not inactivated. However, considering that in raw milk mesophilic spore counts are 2.6-2.9 log cfu/mL, this treatment may be useful.     

Decrease in optical density as a results of germination of Alicyclobacillus acidoterrestris spores under high hydrostatic pressure

Alicyclobacillus acidoterrestris is a spore-forming bacterium, causing spoilage of juices. The spores of these bacteria have the ability to survive in the typical conditions used for thermal pasteurization. Therefore, the use of other techniques such as high hydrostatic pressure is considered for their inactivation. The effect of hydrostatic pressure of 200–500 MPa, at temperatures 4–50 °C for 15 min, on the dynamics of germination of A. acidoterrestris spores in apple juice and pH 4 buffer was studied. To estimate the share of germinated spores, the method of determining the optical density at a wavelength of 660 nm (OD₆₆₀) was used. Parameters of hydrostatic pressure treatment used in this work affected the dynamics of germination of A. acidoterrestris spores in apple juice, and the temperature had the greatest effect. The results indicate that nutrients present in apple juice can promote the germination of A. acidoterrestris spores.     

High pressure inactivation of Clostridium botulinum type E endospores in model emulsion systems

Clostridium botulinum type E is a cold-tolerant, neurotoxigenic, endospore-forming organism, primarily associated with aquatic environments. High pressure thermal (HPT) processing presents a promising tool to enhance food safety and stability. The effect of fat on HPT inactivation of C. botulinum type E spores was investigated using an emulsion model system. The distribution of spores in oil-in-water (O/W) emulsions and their HPT (300–750?MPa, 45–75?°C, 10?min) inactivation was determined as a function of emulsion fat content (30–70% (v/v) soybean oil in buffer). Approximately 26% and 74% of the spores were located at the oil–buffer interface and the continuous phase, respectively. Spore inactivation in emulsion systems decreased with increasing oil contents, which suggests that the fat content of food plays an important role in the protection of C. botulinum type E endospores against HPT treatments. These results can be helpful for future safety considerations.     

Combination of supercritical CO2 and high-power ultrasound for the inactivation of fungal and bacterial spores in lipid emulsions

For the first time, this study addresses the intensification of supercritical carbon dioxide (SC-CO₂) treatments using high-power ultrasound (HPU) for the inactivation of fungal (Aspergillus niger) and bacterial (Clostridium butyricum) spores in oil-in-water emulsions. The inactivation kinetics were analyzed at different pressures (100, 350 and 550 bar) and temperatures (50, 60, 70, 80, 85 °C), depending on the microorganism, and compared to the conventional thermal treatment. The inactivation kinetics were satisfactorily described using the Weibull model.Experimental results showed that SC-CO₂ enhanced the inactivation level of both spores when compared to thermal treatments. Bacterial spores (C. butyricum) were found to be more resistant to SC-CO₂ + HPU, than fungal (A. niger) ones, as also observed in the thermal and SC-CO₂ treatments. The application of HPU intensified the SC-CO₂ inactivation of C. butyricum spores, e.g. shortening the total inactivation time from 10 to 3 min at 85 °C. However, HPU did not affect the SC-CO₂ inactivation of A. niger spores. The study into the effect of a combined SC-CO₂ + HPU treatment has to be necessarily extended to other fungal and bacterial spores, and future studies should elucidate the impact of HPU application on the emulsion’s stability.     

Numerical simulation of thermal and fluiddynamical transport effects on a high pressure induced inactivation

The current paper deals with the effects of convective transport and heterogeneous thermal conditions on the uniformity of a high pressure induced inactivation by means of mathematical modelling and numerical simulation. The inactivation of B. subtilis α-amylase dissolved in TRIS-buffer is chosen as an example for a pressure induced transformation. Two different processes are considered. The first represents a direct treatment where the pressure increase is achieved by mass augmentation of the enzyme solution. The second is an indirect treatment, where the enzyme solution is packed and the pressure increase is achieved by inflow of a pressure medium into the pressure chamber. Aspects of the viscosity of the matrix as well as the heat transfer properties of the packaging material are considered. In both cases, significant process uniformities can be determined.     

Pressure-assisted thermal sterilization of soup

The overall efficiency of an existing scale-up pressure-assisted thermal sterilization (PATS) unit was investigated with regards to inactivation of Geobacillus stearothermophilus spores suspended in pumpkin soup. The PATS unit is a double pipe heat exchanger in which the soup is pumped into its inner high pressure tube and constrained by two high pressure valves, while steam is continuously passed through the annular region to heat the content. The technology is based on pressure generation by thermal expansion of the liquid in an enclosure. In this work, the addition of an air line to push the treated liquid food out of the existing PATS unit has improved the overall quality of the treated samples, as evidenced by achieving higher log reduction of the spores. Compared with thermal processing, the application of PATS shows the potential for lowering the thermal treatment temperature, offering improved food quality.     

High-throughput screening of food additives with synergistic effects on high hydrostatic pressure inactivation of budding yeast

ABSTRACT

We focused on food additives that enhance the effect of high hydrostatic pressure (HHP) treatment for microbial inactivation. We previously isolated Saccharomyces cerevisiae ultraviolet (UV) mutant strain a1210H12 that does not cause a growth delay under specific high pressure treatment conditions. We conceived that it is possible to screen effective food additives in a high-throughput manner by combining strain a1210H12 with a viable cell count method based on liquid culture, named high-throughput microbial pressure inactivation kinetics analysis system (HT-PIKAS). Here, we analyzed the synergistic effect between food additives and HHP treatment on inactivation of strain a1210H12 using HT-PIKAS. We calculated the inactivation rate in the condition of additive only, HHP treatment only and HHP treatment with additive. As a result, four compounds, benzoic acid, sorbic acid, adipic acid and caproic acid, showed high synergistic effects with HHP treatment and could be selected from more than 30 compounds as safe food additives.     

The effect of pressure on bacteriophages

Abstract

Kinetic experiments and electron microscopy have been used to examine pressure induced inactivation of bacteriophages.     

Fluorescence-based methods for the detection of pressure-induced spore germination and inactivation

The application of high pressure (HP) provides an opportunity for the non-thermal preservation of high-quality foods, whereas highly resistant bacterial endospores play an important role. It is known that the germination of spores can be initiated by the application of HP. Moreover, the resistance properties of spores are highly dependent on their physiological states, which are passed through during the germination. To distinguish between different physiological states and to detect the amount of germinated spores after HP treatments, two fluorescence-based methods were applied. A flow cytometric method using a double staining with SYTO 16 as an indicator for germination and propidium iodide as an indicator for membrane damage was used to detect different physiological states of the spores. During the first step of germination, the spore-specific dipicolinic acid (DPA) is released [P. Setlow, Spore germination, Curr. Opin. Microbiol. 6 (2003), pp. 550–556]. DPA reacts with added terbium to form a distinctive fluorescent complex. After measuring the fluorescence intensity at 270 nm excitation wavelength in a fluorescence spectrophotometer, the amount of germinated spores can be determined. Spores of Bacillus subtilis were treated at pressures from 150 to 600 MPa and temperatures from 37 °C to 60 °C in 0.05 M ACES buffer solution (pH 7) for dwell times of up to 2 h. During the HP treatments, inactivation up to 2log ₁₀ cycles and thermal sensitive populations up to 4log ₁₀ cycles could be detected by plate counts. With an increasing number of thermal sensitive spores, an increased proportion of spores in germinated states was detected by flow cytometry. Also the released amount of DPA increased during the dwell times. Moreover, a clear pressure-temperature-time-dependency was shown by screening different conditions. The fluorescence-based measurement of the released DPA can provide the opportunity of an online monitoring of the germination of spores under HP inside the HP vessel. Implementation can be done using diamond anvil cells, units with inspection glasses or by inserting an optical fiber into the HP vessel. The analytical methods used can help to understand the complex mechanism of germination and inactivation of bacterial spores. Due to its universal, process-independent character, the application of these methods is feasible for established and emerging technologies.     

Modification of the radiation sensitivity of anoxic bacterial spores by selected alcohols

The effect of three alcohols as representative of ointment and cream components on the radiation sensitivity of anoxic Bacillus pumilus spores was determined in order to understand the radiation-induced inactivation of spores when suspended in non-aqueous milieux. Bacillus pumilus spores mounted on aluminium silicate powder were suspended in the appropriate alcohol, deoxygenated and ⁶⁰Co gamma-irradiated. Responses ranged between that for buffered suspensions alone to approximately twice that value. Concentrations of the alcohols in buffer, below their solubility limits, did not produce responses significantly different from that characteristic of anoxic buffer alone.     

High Pressure Processing for Preservation,Modification and Transformation of Foods

Commercial, high pressure treated food products have been delivered to the Japanese, the US and the European market. Parallel to these development activities intensive research work has been carried out. Within the European Union three major research projects devoted to high pressure research have been completed and a sound knowledge base has been accumulated on inactivation kinetics of microorganisms and enzymes, on biopolymers (proteins, polysaccharides) as well as on real food systems ( e.g. juices, dairy product, meat, fish, fruits and vegetables). One of the obstacles in furthering high pressure development in Europe is the current Novel Food Legislation and its slow execution.     

Increasing Preservation Efficiency and Product Quality through Control of Temperature Distributions in High Pressure Applications

The effectiveness of HP sterilisation is a function of both temperature and pressure. As during pressurisation the product temperature increases, heat transfer to the colder HPP vessel wall occurs and the product fraction near the vessel wall will be colder than the product in the middle of the vessel. The effect of the temperature distribution in the vessel on the inactivation of Bacillus stearothermophilus has been examined. A mathematical model has been built, in which both thermodynamics and inactivation kinetics are integrated. Heat transfer is based on a Finite Element simulation, inactivation kinetics are based on first order kinetics. Based on this model and experiments the effect of an homogeneous temperature distribution on inactivation is demonstrated.     

热压协同处理对接种于鸡腿菇中的细菌芽孢灭活效果的影响

以鸡腿菇为原料,以凝结芽孢杆菌芽孢和嗜热脂肪芽孢杆菌芽孢为对象菌,研究中温与超高静压协同处理条件下两种对象菌的灭活效果及其相应的动力学变化趋势。研究结果表明:在相同条件下,鸡腿菇中凝结芽孢杆菌芽孢的致死效果优于嗜热脂肪芽孢杆菌;接种于鸡腿菇中的两种对象菌芽孢致死效果均低于磷酸缓冲液中的效果,说明鸡腿菇对这两种细菌芽孢有一定的保护作用。利用线性模型、Weibull模型和Log-logistic模型,对鸡腿菇中两种对象菌芽孢的灭活效果进行拟合,以均方差和回归系数为评价指标,分析了3种模型的拟合效果。结果表明:在所研究的条件下,Log-logistic模型的拟合效果最好,Weibull模型次之,线性模型最差。因此可以通过Log-logistic模型来预测热压协同处理下细菌芽孢的灭活效果。     

Effect of high hydrostatic pressure on enzyme activity in unpasteurized draft sake

ABSTRACT

Unpasteurized draft sake has a potentially high market value, due to its fresh flavor and fruity taste, compared with conventional thermal-pasteurized sake. However, the shelf life of draft sake is limited. To increase the shelf life of draft sake, it is necessary to suppress flavor and taste deterioration resulting from inactivation of enzymes produced by koji-mold. Draft sake was treated with high hydrostatic pressure (HHP) of 200 to 500?MPa at ?7 to 50°C to analyze the inactivation of α-amylases, glucose-forming enzymes, and acid carboxypeptidases. We found significant inactivation of enzymes produced by koji-mold in draft sake subjected to HHP treatment at both high and low temperature. However, HHP treatment at low temperature effectively inactivated enzymes while retaining the fresh flavor and fruity taste of draft sake.     

Possible mechanism of high pressure inactivation of microorganisms

Abstract

We have formulated the hypothesis that the ATPase bound to the cell membrane is strongly involved in high pressure inactivation. The stability of the membrane bound ATPase under pressure would in turn be dependent on the fluidity of the membrane. It has been postulated that cells with a more fluid membrane would be more resistant to pressure. We have confirmed this by comparing cells of Lactobacillus plantarum which differed in fluidity of the membrane. Damage of the membrane by pressure was shown by staining with propidium iodide. Leakage of ATP from the cells was observed when the cells were subjected to pressure, which was also an indication of membrane damage. ATP could not be formed after severe pressure treatment, but could after mild pressure treatment. This is also in line with the above mentioned hypothesis.     

Pressure-Assisted Freezing and Thawing of Foods: A Review of Recent Studies

Phase transition phenomena take place in aqueous solutions, model foods and cellular tissues subjected to combined high pressure and low (subzero) temperature. The kinetics and mechanisms of pressure-shift ice nucleation, of type III, V or VI ice crystal formation, or of pressure-thawing are still poorly documented. Physical and chemical factors affect the ice crystal size distribution throughout sample depth. These phase transition phenomena, as well as the pressure level, influence both chemical constituents (proteins, enzymes) and structural elements (gels, emulsions, cellular tissues, micro-organisms). Recent studies indicate that: (1) in pressure-shift freezing of muscle foods, the aggregation of myofibrillar proteins and the resulting toughness due to high pressure exposition appear to offset the benefit of small ice crystal formation; (2) the cell and tissue structure of some fruits or vegetables is less disrupted by pressure-shift than air-blast freezing, but it is doubtful whether this brings improved appearance, texture or water retention; (3) high pressure-low temperature inactivation of enzymes is not sufficient to replace thermal blanching; (4) high pressure microbial inactivation is enhanced below 0 °C, but probably not enough for practical applications; (5) the benefits of pressure-thawing in terms of enhanced rate and hygiene may not compensate for increased equipment and packaging costs; (6) progress is made in modelling pressure-freezing and thawing, and in assessing the extent of pressure-shift nucleation.     

Virulence of Listeria monocytogenes before and after high hydrostatic pressure treatment

Abstract

High hydrostatic pressure treatment is regarded as a possible alternative process for food preservation. One of the primary considerations for industrial applications is the ability of this technique to destroy pathogenic microorganisms. The inactivation of microorganism populations after high pressure treatment is well described, but their residual pathogenicity is less documented. This study compared the virulence of Listeria monocytogenes scott A before and after high pressure treatments. The pressurized samples were composed of 1 ml of Listeria monocytogenes culture diluted into 9 ml of buffer. Two buffers are used: phosphate buffer (pH 7) and citrate buffer (pH 5.6).

The virulence of cells is estimated by their association and invasion capacities to Hela cells. These characteristics of pathogenity were compared between untreated and pressurized cells (400 MPa, 10min, in citrate buffer) or (600 MPa, 10min, in phosphate buffer). These results showed that, after treatment in citrate buffer, the treated cells lost their association and invasion capacities. However, in a phosphate buffer, treated cells have lose their invasion properties, but retain their association capacities to Hela cells. These results showed that the inactivation of Listeria monocytogenes population involve a loss of their pathogenic properties. However, the conservation of association capacities of some inactivated cells show that these cells seem to continue to express adhesion molecules and, in certain conditions, can reestablish their multiplication properties.     

Effect of dry-ozone exposure on different polymer surfaces and their resulting biocidal action on sporulated bacteria

The current work describes a novel technique by which certain types of polymers subjected to dry gaseous ozone acquire the ability to inactivate microorganisms, including those as resistant as bacterial spores. The originality and advantages of this ozone treatment of polymer surfaces rest on its simplicity (achieved at ambient temperature and pressure, a one step process …) and its efficacy. The inactivation efficiency is found to be specific to the nature of the treated polymer: 24 h after deposition of 10⁶ B. atrophaeus spores from a 100 µL suspension, high inactivation rates are observed with polymethyldisiloxane (99.997%, almost 5 log) and polystyrene (99.7%, more than 2 log), a lower rate with polyurethane (99.1%, 2 log) whereas polytetrafluoroethylene shows no detectable biocidal activity. Changes in hydrophilicity of these surfaces are monitored by means of contact-angle measurements while topographic modifications are characterized through atomic force microscopy. Ozone exposure brings about important topographic changes and chemical modifications on some polymers, which can be correlated with oxidation processes, increased wettability and surface energy. Variations of the dispersive and non-dispersive (polar) components of the surface energy are partially correlated with the polymer biocidal response. Furthermore, the basic component of the treated polymer (in contrast to its acidic component) seems to be correlated with the biocidal activity of the treated surfaces. Chemical species bearing ester groups, probably partially-oxidized styrene oligomers, as revealed by chemical analysis, could be involved in the biocidal activity. On practical grounds, since some of these treated polymers can strongly reduce microorganism loads on their surfaces, they could be particularly useful in hospital environment.