Kai Reineke

Emerging Technologies in Food Processing

High hydrostatic pressure (HHP), pulsed electric fields (PEFs), ultrasound (US), and cold plasma (CP) are emerging technologies that have already found application in the food industry or related sectors. This review aims to describe the basic principles of these nonthermal technologies as well as the state of the art concerning their impact on biological cells, enzymes, and food constituents. Current and potential applications will be discussed, focusing on process-structure-function relationships, as well as recent advances in the process development.

Mechanisms of endospore inactivation under high pressure

It is well known that spore germination and inactivation can be achieved within a broad temperature and pressure range. The existing literature, however, reports contradictory results concerning the effectiveness of different pressure-temperature combinations and the underlying inactivation mechanism(s). Much of the published kinetic data are prone to error as a result of unstable process conditions or an incomplete investigation of the entire inactivation pathway. Here, we review this field of research, and also discuss an inactivation mechanism of at least two steps and propose an inactivation model based on current data. Further, spore resistance properties and matrix interactions are linked to spore inactivation effectiveness.

The impact of high pressure and temperature on bacterial spores: inactivation mechanisms of Bacillus subtilis above 500 MPa.

UNLABELLED High-pressure thermal sterilization (HPTS) is an emerging technology to produce shelf stable low acid foods. Pressures below 300 MPa can induce spore germination by triggering germination receptors. Pressures above 500 MPa could directly induce a Ca+2-dipicolinic acid (DPA) release, which triggers the cortex-lytic enzymes (CLEs). It has been argued that the activated CLEs could be inactivated under HPTS conditions. To test this claim, a wild-type strain and 2 strains of Bacillus subtilis spores lacking germinant receptors and one of 2 CLEs were treated simultaneously from 550 to 700 MPa and 37 to 80 C (slow compression) and at 60 to 80 C up to 1 GPa (fast compression). Besides, an additional heat treatment to determine the amount of germinated cells, we added TbCl3 to detect the amount of DPA released from the spore core via fluorescent measurement. After pressure treatment for 120 min at 550 MPa and 37 °C, no inactivation was observed for the wild-type strain. The amount of released DPA correlated to the amount of germinated spores, but always higher compared to the belonging cell count after pressure treatment. The release of DPA and the increase of heat-sensitive spores confirm that the inactivation mechanism during HPTS passes through the physiological states: (1) dormancy, (2) activation, and (3) inactivation. As the intensity of treatment increased, inactivation of all spore strains also strongly increased (up to -5.7 log10), and we found only a slight increase in the inactivation of one of the CLE (sleB). Furthermore, above a certain threshold pressure, temperature became the dominant influence on germination rate. PRACTICAL APPLICATION   The continuous increase of high-pressure (HP) research over the last several decades has already generated an impressive number of commercially available HP pasteurized products. Furthermore, research helped to provoke the certification of a pressure-assisted thermal sterilization process by the U.S. FDA in February 2009. However, this promising sterilization technology has not yet been applied in industrial settings. An improved understanding of spore inactivation mechanisms and the ability to calculate desired inactivation levels will help to make this technology available for pilot studies and commercialization at an industrial scale. Moreover, if the synergy between pressure and elevated temperature on the inactivation rate could be identified, clarification of the underlying inactivation mechanism during HP thermal sterilization could help to further optimize the process of this emerging technology.

High pressure thermal sterilization – development and application of temperature controlled spore inactivation studies

High pressure thermal sterilization is an emerging technology that can produce uniform, minimally processed foods of high quality, better than heat treatment alone. At present, it has not yet been successfully introduced into the food industry, possibly due to the less known inactivation mechanism of high resistant bacterial spores. This study developed and used a new analytical tool to improve the understanding of spore mechanisms at high pressures and temperatures. The generated data were exemplarily incorporated into analyses of industrial sterilization processes. An improved understanding of the mechanisms of spore inactivation will aid in the food safety assessment of high pressure thermal sterilization in particular, and also assist in the commercialization of this novel process, facilitating adoption by industry.

High‐Pressure Thermal Sterilization: Food Safety and Food Quality of Baby Food Puree

The benefits that high-pressure thermal sterilization offers as an emerging technology could be used to produce a better overall food quality. Due to shorter dwell times and lower thermal load applied to the product in comparison to the thermal retorting, lower numbers and quantities of unwanted food processing contaminants (FPCs), for example, furan, acrylamide, HMF, and MCPD-esters could be formed. Two spore strains were used to test the technique; Geobacillus stearothermophilus and Bacillus amyloliquefaciens, over the temperature range 90 to 121 °C at 600 MPa. The treatments were carried out in baby food puree and ACES-buffer. The treatments at 90 and 105 °C showed that G. stearothermophilus is more pressure-sensitive than B. amyloliquefaciens. The formation of FPCs was monitored during the sterilization process and compared to the amounts found in retorted samples of the same food. The amounts of furan could be reduced between 81% to 96% in comparison to retorting for the tested temperature pressure combination even at sterilization conditions of F₀-value in 7 min.

The release of dipicolinic acid--the rate-limiting step of Bacillus endospore inactivation during the high pressure thermal sterilization process.

High pressure combined with elevated temperatures can produce low acid, commercially sterile and shelf-stable foods. Depending on the temperature and pressure levels applied, bacterial endospores pass through different pathways, which can lead to a pressure-induced germination or inactivation. Regardless of the pathway, Bacillus endospores first release pyridine-2,6-dicarboxylic acid (DPA), which contributes to the low amount of free water in the spore core and is consequently responsible for the spores high resistance against wet and dry heat. This is therefore the rate-limiting step in the high pressure sterilization process. To evaluate the impact of a broad pressure, temperature and time domain on the DPA release, Bacillus subtilis spores were pressure treated between 0.1 and 900 MPa at between 30 and 80 °C under isothermal isobaric conditions during dwell time. DPA quantification was assessed using HPLC, and samples were taken both immediately and 2 h after the pressure treatment. To obtain a release kinetic for some pressure-temperature conditions, samples were collected between 1s and 60 min after decompression. A multiresponse kinetic model was then used to derive a model covering all kinetic data. The isorate lines modeled for the DPA release in the chosen pressure-temperature landscape enabled the determination of three distinct zones. (I) For pressures <600 MPa and temperatures >50 °C, a 90% DPA release was achievable in less than 5 min and no difference in the amount of DPA was found immediately 2 h after pressurization. This may indicate irreversible damage to the inner spore membrane or membrane proteins. (II) Above 600 MPa the synergism between pressure and temperature diminished, and the treatment temperature alone dominated DPA release. (III) Pressures <600 MPa and temperatures <50 °C resulted in a retarded release of DPA, with strong increased differences in the amount of DPA released after 2 h, which implies a pressure-induced physiological like germination with cortex degradation, which continues after pressure release. Furthermore, at 600 MPa and 40 °C, a linear relationship was found for the DPA release rate constants ln(k(DPA)) between 1 and 30 min.

Sterilization of liquid foods by pulsed electric fields-an innovative ultra-high temperature process

The intention of this study was to investigate the inactivation of endospores by a combined thermal and pulsed electric field (PEF) treatment. Therefore, self-cultivated spores of Bacillus subtilis and commercial Geobacillus stearothermophilus spores with certified heat resistance were utilized. Spores of both strains were suspended in saline water (5.3 mS cm−1), skim milk (0.3% fat; 5.3 mS cm−1) and fresh prepared carrot juice (7.73 mS cm−1). The combination of moderate preheating (70–90°C) and an insulated PEF-chamber, combined with a holding tube (65 cm) and a heat exchanger for cooling, enabled a rapid heat up to 105–140°C (measured above the PEF chamber) within 92.2–368.9 μs. To compare the PEF process with a pure thermal inactivation, each spore suspension was heat treated in thin glass capillaries and D-values from 90 to 130°C and its corresponding z-values were calculated. For a comparison of the inactivation data, F-values for the temperature fields of both processes were calculated by using computational fluid dynamics (CFD). A preheating of saline water to 70°C with a flow rate of 5 l h−1, a frequency of 150 Hz and an energy input of 226.5 kJ kg−1, resulted in a measured outlet temperature of 117°C and a 4.67 log10 inactivation of B. subtilis. The thermal process with identical F-value caused only a 3.71 log10 inactivation. This synergism of moderate preheating and PEF was even more pronounced for G. stearothermophilus spores in saline water. A preheating to 95°C and an energy input of 144 kJ kg−1 resulted in an outlet temperature of 126°C and a 3.28 log10 inactivation, whereas nearly no inactivation (0.2 log10) was achieved during the thermal treatment. Hence, the PEF technology was evaluated as an alternative ultra-high temperature process. However, for an industrial scale application of this process for sterilization, optimization of the treatment chamber design is needed to reduce the occurring inhomogeneous temperature fields.

High pressure thermal inactivation of Clostridium botulinum type E endospores – kinetic modeling and mechanistic insights

Cold-tolerant, neurotoxigenic, endospore forming Clostridium (C.) botulinum type E belongs to the non-proteolytic physiological C. botulinum group II, is primarily associated with aquatic environments, and presents a safety risk for seafood. High pressure thermal (HPT) processing exploiting the synergistic effect of pressure and temperature can be used to inactivate bacterial endospores. We investigated the inactivation of C. botulinum type E spores by (near) isothermal HPT treatments at 300–1200 MPa at 30–75°C for 1 s to 10 min. The occurrence of heat and lysozyme susceptible spore fractions after such treatments was determined. The experimental data were modeled to obtain kinetic parameters and represented graphically by isoeffect lines. In contrast to findings for spores of other species and within the range of treatment parameters applied, zones of spore stabilization (lower inactivation than heat treatments alone), large heat susceptible (HPT-induced germinated) or lysozyme-dependently germinable (damaged coat layer) spore fractions were not detected. Inactivation followed first order kinetics. Dipicolinic acid release kinetics allowed for insights into possible inactivation mechanisms suggesting a (poorly effective) physiologic-like (similar to nutrient-induced) germination at ≤450 MPa/≤45°C and non-physiological germination at >500 MPa/>60–70°C. Results of this study support the existence of some commonalities in the HPT inactivation mechanism of C. botulinum type E spores and Bacillus spores although both organisms have significantly different HPT resistance properties. The information presented here contributes to closing the gap in knowledge regarding the HPT inactivation of spore formers relevant to food safety and may help industrial implementation of HPT processing. The markedly lower HPT resistance of C. botulinum type E spores compared with the resistance of spores from other C. botulinum types could allow for the implementation of milder processes without endangering food safety.

Impact of surface structure and feed gas composition on Bacillus subtilis endospore inactivation during direct plasma treatment

This study investigated the inactivation efficiency of cold atmospheric pressure plasma treatment on Bacillus subtilis endospores dependent on the used feed gas composition and on the surface, the endospores were attached on. Glass petri-dishes, glass beads, and peppercorns were inoculated with the same endospore density and treated with a radio frequency plasma jet. Generated reactive species were detected using optical emission spectroscopy. A quantitative polymerase chain reaction (qPCR) based ratio detection system was established to monitor the DNA damage during the plasma treatment. Argon + 0.135% vol. oxygen + 0.2% vol. nitrogen as feed gas emitted the highest amounts of UV-C photons and considerable amount of reactive oxygen and nitrogen species. Plasma generated with argon + 0.135% vol. oxygen was characterized by the highest emission of reactive oxygen species (ROS), whereas the UV-C emission was negligible. The use of pure argon showed a negligible emission of UV photons and atomic oxygen, however, the emission of vacuum (V)UV photons was assumed. Similar maximum inactivation results were achieved for the three feed gas compositions. The surface structure had a significant impact on the inactivation efficiency of the plasma treatment. The maximum inactivation achieved was between 2.4 and 2.8 log10 on glass petri-dishes and 3.9 to 4.6 log10 on glass beads. The treatment of peppercorns resulted in an inactivation lower than 1.0 log10. qPCR results showed a significant DNA damage for all gas compositions. Pure argon showed the highest results for the DNA damage ratio values, followed by argon + 0.135% vol. oxygen + 0.2% vol. nitrogen. In case of argon + 0.135% vol. oxygen the inactivation seems to be dominated by the action of ROS. These findings indicate the significant role of VUV and UV photons in the inactivation process of B. subtilis endospores.

Shift of pH-Value During Thermal Treatments in Buffer Solutions and Selected Foods

The pH value is one of the most important process parameters during thermal treatments, regardless if the medium is a simple buffer solution or a complex food matrix. When the temperature increases after an initial measurement of the pH at ambient temperature (25°C), a significant pH shift could occur, which could produce incomparable results in different buffer solutions or lead to side reactions during food preservation. Consequently, a measurement cell was constructed to record online the pH-value and temperature up to 130°C. By applying the Nernst equation, it was possible to exclude the temperature-dependent influence of the pH glass electrode on the total pH value. The pH shift was measured over a wide temperature range (ΔT 20–130°C) in the most commonly used buffer solutions and some selected food matrices. The ΔpH of certain buffer solutions, namely TRIS and ACES, showed a significant pH decrease of −2.01 ± 0.08 (ΔT 20–130°C) and −1.27 ± 0.1 (ΔT 20–130°C), respectively, whereas the pH of PBS buffer solution was nearly independent of temperature. The ΔpH decrease recorded in milk (−0.89 ± 0.6, ΔT 20–130°C) as well as commercial and self-made baby food (−0.56 ± 0.05, ΔT 20–130°C) is of special interest for the food industry to get a deeper insight in occurring reactions during thermal preservation processes.