High pressure CO2 reduces the wet heat resistance of Bacillus subtilis spores by perturbing the inner membrane

Spores of wild-type Bacillus subtilis PS533 were treated by wet heat at 75 °C for 30 min, and high pressure CO₂ (HPCD) at 6.5 MPa and 30 °C or 75 °C for 30 min. The spores were analyzed for wet heat resistance (85 °C, 90 °C, 95 °C) and typical germination events including DPA release and cortex hydrolysis, inner membrane permeability, and germination triggered by nutrient (L-valine and AGFK) or non-nutrient (dodecylamine and high pressure at 150 MPa or 550 MPa) germinants. The results showed that (i) HPCD-treated spores exhibited reduced wet heat resistance compared to the untreated or wet heat-treated spores; (ii) HPCD-treated spores did not undergo typical germination events such as DPA release or cortex hydrolysis compared to normally germinated spores; (iii) HPCD-treated spores released more metal ions and exhibited decreased ability to maintain DPA, indicating that the permeability of inner membrane of HPCD-treated spores was increased; (iv) HPCD-treated spores exhibited reduced germination rate when triggered by L-valine or 150 MPa, but increased germination rate when triggered by dodecylamine or 550 MPa, suggesting that the fluidity of the inner membrane of HPCD-treated spores might be increased. These results indicated that HPCD could reduce the wet heat resistance of spores, and this resistance decrease was probably due to the modification of the inner membrane caused by HPCD.Industrial relevanceThe extremely high wet heat resistance of spores makes them a significant problem in the thermal processing of foods. Thus, it of great interest to develop a process to reduce the wet heat resistance of spores. In this work, we found that HPCD can significantly reduce the wet heat resistance of B. subtilis spores, and this was achieved by perturbing the inner membrane of spores. These results can improve our understanding of the inactivation mechanism of spores by HPCD, and also provide an alternative approach for spore inactivation in foods.     

Inhibition of nutrient- and high pressure-induced germination of Bacillus cereus spores by plant essential oils

The efficiency of high-pressure (HP) treatment to eliminate vegetative bacterial cells is synergistically increased by many natural antimicrobials, but the effects on spores are poorly described. Here we report the effect of eleven plant essential oils on the nutrient- and HP-induced germination of spores of a group VI psychrotolerant Bacillus cereus strain. Ten oils partially inhibited nutrient-induced germination. These oils also inhibited HP-induced germination, but some inhibited only germination at moderate (200 MPa) pressure and others only at very high (600 MPa) pressure. Inhibition of spore germination by essential oils may have an adverse effect on the effectiveness of spore inactivation by HP at moderate temperatures, and this should be taken into account when designing combined processes. Essential oil from carrot seed did not inhibit nutrient or HP germination although it showed growth inhibitory properties, and essential oils with these properties may therefore open interesting perspectives in combination treatments with HP.Industrial relevanceHP treatment is an alternative processing technique that preserves a better balance of food quality and microbiological safety as compared to thermal processing. While most vegetative bacteria are efficiently inactivated by HP, inactivation of spores is inefficient. At moderate temperature, spore inactivation proceeds in a two-step process in which spores first germinate and are subsequently inactivated. The combination with natural antimicrobials is a promising approach to enhance the efficiency of HP processing because it exerts a synergistic effect on inactivation of vegetative bacteria. However, the current work is one of the first to document the effect of essential oils on the HP-induced germination of spores.     

HIGH PRESSURE–HIGH TEMPERATURE STERILIZATION: FROM KINETIC ANALYSIS TO PROCESS VERIFICATION†

The inactivation of Clostridium sporogenes PA 3679 spores by high pressure at high temperatures (HP–HT) in phosphate buffer was investigated in a lab‐scale temperature‐controlled HP system (QFP‐6) with an internal heater to maintain the sample temperature. Some inactivation of spores occurred during the pressurization come‐up time (CUT) and depressurization time. The inactivation of PA 3679 was found to be exponential during the adiabatic holding period of the HP cycle at constant pressures and temperatures. The inactivation rate increased with both pressure and temperature. The kinetic parameters – such as D‐values at tested temperatures and pressures that are necessary for the design of process parameters of HP sterilization process – were determined. Within the pressure range of 600–800 MPa, the calculated D‐values ranged from 270.3 to 357.4 and 49.0 to 67.6 s at 91 and 108C, respectively. These studies provided basic data on the effects of pressure and temperature on the inactivation of PA 3679 spores under conditions applicable to the development of preservation specifications for commercial HP–HT processing of low acid foods. The spore strips of C. sporogenes were used as indicators for microbiological verification of delivered lethality of HP–HT sterilization process at different processing conditions in a pilot scale HP vessel.     

Inactivation of Bacillus cereus spores in milk by mild pressure and heat treatments

The objective of this work was to study the germination and subsequent inactivation of Bacillus cereus spores in milk by mild hydrostatic pressure treatment. In an introductory experiment with strain LMG6910 treated at 40 degrees C for 30 min at 0, 100, 300 and 600 MPa, germination levels were 1.5 to 3 logs higher in milk than in 100 mM potassium phosphate buffer (pH 6.7). The effects of pressure and germination-inducing components present in the milk on spore germination were synergistic. More detailed experiments were conducted in milk at a range of pressures between 100 and 600 MPa at temperatures between 30 and 60 degrees C to identify treatments that allow a 6 log inactivation of B. cereus spores. The mildest treatment resulting in a 6 log germination was 30 min at 200 MPa/40 degrees C. Lower treatment pressures or temperatures resulted in considerably less germination, and higher pressures and temperatures further increased germination, but a small fraction of spores always remained ungerminated. Further, not all germinated spores were inactivated by the pressure treatment, even under the most severe conditions (600 MPa/60 degrees C). Two possible approaches to achieve a 6 log spore inactivation were identified, and validated in three additional B. cereus strains. The first is a single step treatment at 500 MPa/60 degrees C for 30 min, the second is a two-step treatment consisting of pressure treatment for 30 min at 200 MPa/45 degrees C to induce spore germination, followed by mild heat treatment at 60 degrees C for 10 min to kill the germinated spores. Reduction of the pressurization time to 15 min still allows a 5 log inactivation. These results illustrate the potential of high-pressure treatment to inactivate bacterial spores in minimally processed foods.     

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

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

Effect of small, acid-soluble proteins on spore resistance and germination under a combination of pressure and heat treatment

To find the range of pressure required for effective high-pressure inactivation of bacterial spores and to investigate the role of alpha/beta-type small, acid-soluble proteins (SASP) in spores under pressure treatment, mild heat was combined with pressure (room temperature to 65 degrees C and 100 to 500 MPa) and applied to wild-type and SASP-alpha-/beta- Bacillus subtilis spores. On the one hand, more than 4 log units of wild-type spores were reduced after pressurization at 100 to 500 MPa and 65 degrees C. On the other hand, the number of surviving mutant spores decreased by 2 log units at 100 MPa and by more than 5 log units at 500 MPa. At 500 MPa and 65 degrees C, both wild-type and mutant spore survivor counts were reduced by 5 log units. Interestingly, pressures of 100, 200, and 300 MPa at 65 degrees C inactivated wild-type SASP-alpha+/beta+ spores more than mutant SASP-alpha-/beta- spores, and this was attributed to less pressure-induced germination in SASP-alpha-/beta- spores than in wild-type SASP-alpha+/beta+ spores. However, there was no difference in the pressure resistance between SASP-alpha+/beta+ and SASP-alpha-/beta- spores at 100 MPa and ambient temperature (approximately 22 degrees C) for 30 min. A combination of high pressure and high temperature is very effective for inducing spore germination, and then inactivation of the germinated spore occurs because of the heat treatment. This study showed that alpha/beta-type SASP play a role in spore inactivation by increasing spore germination under 100 to 300 MPa at high temperature.     

Germination and inactivation of Bacillus coagulans and Alicyclobacillus acidoterrestris spores by high hydrostatic pressure treatment in buffer and tomato sauce

Acidothermophilic bacteria like Alicyclobacillus acidoterrestris and Bacillus coagulans can cause spoilage of heat-processed acidic foods because they form spores with very high heat resistance and can grow at low pH. The objective of this work was to study the germination and inactivation of A. acidoterrestris and B. coagulans spores by high hydrostatic pressure (HP) treatment at temperatures up to 60 °C and both at low and neutral pH. In a first experiment, spores suspended in buffers at pH 4.0, 5.0 and 7.0 were processed for 10 min at different pressures (100-800 MPa) at 40 °C. None of these treatments caused any significant inactivation, except perhaps at 800 MPa in pH 4.0 buffer where close to 1 log inactivation of B. coagulans was observed. Spore germination up to about 2 log was observed for both bacteria but occurred mainly in a low pressure window (100-300 MPa) for A. acidoterrestris and only in a high pressure window (600-800 MPa) for B. coagulans. In addition, low pH suppressed germination in A. acidoterrestris, but stimulated it in B. coagulans. In a second series of experiments, spores were treated in tomato sauce of pH 4.2 and 5.0 at 100 - 800 MPa at 25, 40 and 60 °C for 10 min. At 40 °C, results for B. coagulans were similar as in buffer. For A. acidoterrestris, germination levels in tomato sauce were generally higher than in buffer, and showed little difference at low and high pressure. Remarkably, the pH dependence of A. acidoterrestris spore germination was reversed in tomato sauce, with more germination at the lowest pH. Furthermore, HP treatments in the pH 4.2 sauce caused between 1 and 1.5 log inactivation of A. acidoterrestris. Germination of spores in the high pressure window was strongly temperature dependent, whereas germination of A. acidoterrestris in the low pressure window showed little temperature dependence. When HP treatment was conducted at 60 °C, most of the germinated spores were also inactivated. For the pH 4.2 tomato sauce, this resulted in up to 3.5 and 2.0 log inactivation for A. acidoterrestris and B. coagulans respectively. We conclude that HP treatment can induce germination and inactivation of spores from thermoacidophilic bacteria in acidic foods, and may thus be useful to reduce spoilage of such foods caused by these bacteria.     

Bacillus spore germination at moderate high pressure: A review on underlying mechanisms,influencing factors,and its comparison with nutrient germination

Spore-forming bacteria are resistant to stress conditions owing to their ability to form highly resistant dormant spores. These spores can survive adverse environmental conditions in nature, as well as decontamination processes in the food and related industries. Bacterial spores may return to their vegetative state through a process called germination. As spore germination is critical for the loss of resistance, outgrowth, and development of pathogenicity and spoilage potential, the germination pathway has piqued the interest of the scientific community. The inhibition and induction of germination have critical applications in the food industry. Targeted germination can aid in decreasing the resistance of spores and allow the application of milder inactivation procedures. This germination-inactivation strategy allows better maintenance of important food quality attributes. Different stimuli are reported to trigger germination. Among those, isostatic high pressure (HP) has gained increasing attention due to its potential applications in industrial processes. However, pressure-mediated spore germination is extremely heterogeneous as some spores germinate rapidly, while others exhibit slow germination or do not undergo germination at all. The successful and safe implementation of the germination-inactivation strategy, however, depends on the germination of all spores. Therefore, there is a need to elucidate the mechanisms of HP-mediated germination. This work aimed to critically review the current state of knowledge on Bacillus spore germination at a moderate HP of 50–300 MPa. In this review, the germination mechanism, heterogeneity, and influencing factors have been outlined along with knowledge gaps.     

Continuous Versus Discontinuous Ultra‐High‐Pressure Systems for Food Sterilization with Focus on Ultra‐High‐Pressure Homogenization and High‐Pressure Thermal Sterilization: A Review

High‐pressure thermal sterilization (HPTS) and ultra‐high‐pressure homogenization (UHPH) are two emerging sterilization techniques that have not been implemented in the food industry yet. The two technologies apply different acting principles as HPTS uses isostatic pressure in combination with heat whereas UHPH uses dynamic pressure in combination with shear stress, cavitation, impingement, and heat. Both technologies offer significant benefits in terms of spore inactivation in food production with reduced thermal intensity and minimized effects on sensory and nutritional profiles. These benefits have resulted in relevant research efforts on both technologies over the past few decades. This state of the art of the discontinuous HPTS‐based and the continuous UHPH‐based sterilization concepts are assessed within this review. Further, various basic principles and promising future preservation applications of HPTS and UHPH for food processing, that are also applicable in the pharmaceutical, biochemical, and biotechnological sectors, are summarized. In addition, the applications and limitations of these technologies in terms of optimizations needed to overcome the identified challenges are emphasized.     

Effect of high pressure thermal sterilization on the formation of food processing contaminants

The use of high pressure thermal sterilization (HPTS) as an emerging technology for the sterilization of foods could be a big turning point in the food industry. HPTS can result in a better overall food quality, lower thermal load applied to the product and less unwanted food processing contaminants (FPCs) as e.g. furan and monochloropropanediol/-esters.Hence, within the EU FP7 founded Prometheus-project HPTS treatments were performed for selected food systems. Therefore, two spore strains were tested, the Geobacillus stearothermophilus and the Bacillus amyloliquefaciens, in the temperature range from 90 to 121 °C at 600 MPa. The treatments were carried out in different fish system and ACES-buffer. The treatment at 90 and 105 °C showed that the G. stearothermophilus is more pressure sensitive than the 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 systems. Depending on the food system the amounts of furan could be reduced between 71 and 97% for the tested temperature pressure combination even at sterilization conditions of F₀-value 7 min.Industrial relevanceThe high pressure thermal sterilization (HPTS) process is an emerging technology to produce high quality low acid food products, which are shelf-stable at ambient temperature. In addition the consumer today demands foods which are minimally processed and are healthy and safe. However, an industrial scale process has not yet been implemented.The work in this paper shows different temperature combinations (90 to 121 °C) at 600 MPa and their influence on the endospore inactivation and also on the formation of unwanted food process contaminants (FPCs), such as furan and monochloropropanediol/-esters, in comparison to retorting. The use of HPTS could lead to shorter process times and a means by which a better quality of the foods could be achieved.     

Activation and inactivation of Talaromyces macrosporus ascospores by high hydrostatic pressure

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

Strategy to inactivate Clostridium perfringens spores in meat products

The current study aimed to develop an inactivation strategy for Clostridium perfringens spores in meat through a combination of spore activation at low pressure (100–200 MPa, 7 min) and elevated temperature (80 °C, 10 min); spore germination at high temperatures (55, 60 or 65 °C); and inactivation of germinated spores with elevated temperatures (80 and 90 °C, 10 and 20 min) and high pressure (586 MPa, at 23 and 73 °C, 10 min). Low pressures (100–200 MPa) were insufficient to efficiently activate C. perfringens spores for germination. However, C. perfringens spores were efficiently activated with elevated temperature (80 °C, 10 min), and germinated at temperatures lethal for vegetative cells (≥55 °C) when incubated for 60 min with a mixture of l-asparagine and KCl (AK) in phosphate buffer (pH 7) and in poultry meat. Inactivation of spores (∼4 decimal reduction) in meat by elevated temperatures (80–90 °C for 20 min) required a long germination period (55 °C for 60 min). However, similar inactivation level was reached with shorter germination period (55 °C for 15 min) when spore contaminated-meat was treated with pressure-assisted thermal processing (568 MPa, 73 °C, 10 min). Therefore, the most efficient strategy to inactivate C. perfringens spores in poultry meat containing 50 mM AK consisted: (i) a primary heat treatment (80 °C, 10 min) to pasteurize and denature the meat proteins and to activate C. perfringens spores for germination; (ii) cooling of the product to 55 °C in about 20 min and further incubation at 55 °C for about 15 min for spore germination; and (iii) inactivation of germinated spores by pressure-assisted thermal processing (586 MPa at 73 °C for 10 min). Collectively, this study demonstrates the feasibility of an alternative and novel strategy to inactivate C. perfringens spores in meat products formulated with germinants specific for C. perfringens.     

高压热杀菌处理对枯草杆菌芽孢皮层裂解酶活力的影响

高压热杀菌处理(HPTS)下芽孢皮层肽聚糖水解是造成细菌芽孢死亡的重要原因,芽孢自身皮层裂解酶活力变化对皮层肽聚糖水解有直接影响。本文从枯草杆菌芽孢中提取了皮层裂解酶,先研究了磷酸钠浓度、pH、温度和压力对皮层裂解酶活力的影响,在此基础上研究了HPTS对该酶活力的影响。结果发现,皮层裂解酶的最适磷酸钠浓度为0.05 mol/L,当磷酸钠浓度为0.35 mol/L 时,皮层裂解酶活力较低;当pH为7时,皮层裂解酶活力最强,偏酸或偏碱性条件下,活力均有所降低;在4~44 ℃时皮层裂解酶均具活力,酶活变化无明显规律,但当温度升至68 ℃时皮层裂解酶基本失活;在150~530 MPa压力范围内,压力对皮层裂解酶活性基本无影响,然而HPTS处理时(530 MPa、68 ℃、15 min),皮层裂解酶基本失活,而皮层裂解酶是芽孢中唯一能特异性地水解皮层肽聚糖的酶,由此可推测HPTS处理下皮层肽聚糖发生了非酶水解。     

Response of Spores to High‐Pressure Processing

ABSTRACT: This review focuses on the responses of microbial spores to food processes that incorporate high hydrostatic pressures. While the majority of information deals with spores of Bacillus species, spores of Clostridium and Alicyclobacillus species are also discussed, and a section of the review surveys the responses of fungal spores to high‐pressure processing. The mechanisms of the germination of bacterial spores are outlined in detail with regard to spore physiology and structure, along with molecular aspects of germinants and the interaction with spore receptors. Use of treatments combining pressure and temperature for a range of different food products is reviewed, including examples of hurdle technology employing high hydrostatic pressure. Pressure‐assisted thermal sterilization is also discussed.     

非热杀菌技术杀灭食品中芽孢效能及机理研究进展

传统热杀菌会对食品品质产生不利影响,造成食品颜色变化、产生异味、营养损失等不良后果;非热杀菌技术是食品工业新型加工技术,处理过程中可以保持相对较低的温度,对食品的色、香、味以及营养成分影响较小;同时有利于保持食品中各种功能成分的生理活性,可以满足消费者对高品质食品的要求。芽孢在加工过程中抗性强,在食品中萌发和生长的潜力较大,因此,利用低热或非热灭菌技术对芽孢进行灭活是当前食品工业面临的严峻挑战和重要课题。本文综述现有非热杀菌技术（如高静压技术、高压CO2技术、低温等离子体技术、紫外辐射技术、高压脉冲电场技术等）独立处理或与其他处理技术相结合对芽孢灭活的效果及其机理,着重讨论其在食品行业中的应用以及芽孢灭活的分子机制,以期为生产安全食品、减少不同种类食品中微生物污染提供解决方案。     

Compression Heating and Temperature Control for High-Pressure Destruction of Bacterial Spores: An Experimental Method for Kinetics Evaluation

High-pressure (HP) processing is considered as an alternative technique for thermal sterilization of high quality foods. Adiabatic compression during pressurization allows for quick increase in temperature of food products, which is reversed when the pressure is released, thereby providing rapid heating and cooling conditions and hence short process times. However, during the pressure holding time, the product experiences a temperature drop as a result of heat loss to the vessel. The temperature variation during the process and the synergistic effect of temperature and pressure make it difficult to get the required accurate data on microbial spore destruction kinetics. In this study, a polyoxymethylene (POM)-insulated chamber was evaluated for temperature control in the test sample during pressure treatment. Temperature variations in the HP system were measured in milk test samples inside the POM insulator and pressure medium in the HP vessel under various conditions of pressures (500–900 MPa) and initial temperatures (20–80 °C). Results demonstrated that the POM chamber had good thermal-insulation characteristics under pressure and was able to maintain stable operating conditions for microbial spore destruction kinetics. Based on the measured adiabatic temperature change, the required initial temperatures for the test sample and pressure medium were generated as a quadratic function of pressure and temperature. The setup was then verified for pressure inactivation of Clostridium sporogenes (PA 3679) spores in ultra-heat-treated milk. The better temperature stability of test samples during treatment provided a means to gather accurate data on HP destruction kinetics of the microbial spores.     

Cycling versus Continuous High Pressure treatments at moderate temperatures: Effect on bacterial spores?

The advantage of using high pressure (HP) cycling treatment compared with continuous HP treatment was investigated for the inactivation of bacterial spores. The effects of parameters such as pulse number, pressure level, treatment temperature, compression and decompression rates, and time between pulses were evaluated. For this purpose, Bacillus subtilis and B. cereus spores (10⁸ and 10⁶ CFU/mL respectively) were suspended in 2-(N-morpholino) ethanesulfonic acid (MES) buffer solution, tryptone salt (TS) buffer solution, or infant milk and treated by HP cycling at 300–400 MPa, at 38–60 °C, for 1–5 pulses. Pressure cycling reduced the number of viable spores by 1.8 and 5.9 log respectively for B. subtilis and B. cereus species. Continuous HP treatments were performed at the same pressure and temperature for similar treatment durations. Our results showed that the spore inactivation ratio was correlated with the cumulative exposure time to pressure rather than to effects of the cycling process. Greater spore inactivation caused by HP cycling was observed only when faster compression and decompression rates were applied, probably due to adiabatic heating. A three-step kinetic model was developed, which seemed to support our hypothesis regarding the mechanisms of inactivation by pressure cycling and continuous HP treatments.Industrial relevanceThe resistance of bacterial spores to HP limits the industrial applications to refrigerated food products. In this study, we investigated the use of pressure cycling as a means to improve spore baroinactivation at moderate temperatures (T < 60 °C). We showed that cycling pressure does not significantly increase bacterial spore inactivation in comparable treatment duration, but certainly increases material fatigue in HP vessels. Thus, under moderate temperature, cycling pressure treatment is not industrially relevant.     

A study on the effects of high pressure and heat on Bacillus subtilis spores at low pH

Bacillus subtilis spore suspensions were subjected to pressure treatments at 100 and 600 MPa at 40 degrees C and over a pH range from 3 to 8. Inactivation of spores under these conditions was maximally 80% and was not increased at low pH. However, higher levels of inactivation were obtained when spores were first pressure treated at neutral pH and then exposed for 1 h to low pH. This large difference in inactivation could be explained by the finding that pressure-induced spore germination, which is known to occur at neutral pH, was inhibited at low pH (< 5). Pressure treatment at low pH made spores more sensitive to heat inactivation, suggesting that demineralized H-spores had been formed. Changes in spore core hydration and pH upon exposure of spores at low pH were studied in a more direct way using green fluorescent protein expressed in recombinant B. subtilis as a reporter protein, and it was confirmed that pressure and heat increase spore permeability for protons. Based on these results, the potential of low temperature, high pressure processes for spore inactivation in acid products is discussed.     

Inactivation of Bacillus cereus spores using a combined treatment of UV-TiO2 photocatalysis and high hydrostatic pressure

Bacillus cereus spores are resistant to high hydrostatic pressure (HHP) processing treatment. A combination of UV-TiO₂ photocatalysis (UVTP for 10 min) and two cycles of 600 MPa HHP treatment for 10 min for the first cycle and 1 min for the second cycle (UVTP-2HHP) at ambient temperature was applied to inactivate B. cereus spores inoculated on a solidified agar matrix (SAM) used as a model matrix. Two cycles of HHP treatment were used as a strategy for induction of spore germination, followed by inactivation. UVTP and 2 cycles of HHP resulted in a 5.0-log CFU/cm² spore reduction (initial spore count was 6.6 log CFU/cm²), including an approximate 0.8-log CFU/cm² reduction due to a synergistic effect. The inactivation mechanism of UVTP pretreatment was related to lipid peroxidation of the spore membrane based on the level of malondialdehyde (MDA) making spores susceptible to the HHP treatment. Flow cytometry and transmission electron microscopic (TEM) analyses showed severe physiological alteration and structural damage to spores after the combined treatment. UVTP and 2 cycles of HHP showed potential for effective inactivation of B. cereus to ensure food safety from B. cereus spores on food products.Practical applicationsInactivation of bacterial spores remains a technical challenge for HHP and other interventions because spores are highly resistant to high pressure. Pretreatment with UVTP followed by two cycles of HHP resulted in reduction in B. cereus spores due to a synergistic effect. This hurdle technology of UVTP and HHP can help food industry in ensuring food safety against the Bacillus spores.     

Inactivation of Bacillus cereus spores by high hydrostatic pressure at different temperatures

The effect of pH on the initiation of germination and on the inactivation of Bacillus cereus (KCTC 1012) spores during high hydrostatic pressure processing (HPP) with pressures of 0.1 to 600 MPa at different temperatures was investigated. Two different high-pressure treatments were adopted to evaluate the effect of pH on the inactivation of B. cereus on sporulation medium and in suspension medium. Inactivation of B. cereus spores with HPP treatment was affected more by sporulation medium pH than by suspension medium pH. B. cereus spores obtained through sporulation at pH 6.0 showed more resistance to inactivation by HPP at 20, 40, and 60 degrees C than did those obtained through sporulation at pHs of 7.0 and 8.0. Constituents of B. cereus spores obtained through sporulation at pH 6.0 may undergo electrochemical charge changes comparable to those for spores obtained through sporulation at pH 7.0. The initiation of B. cereus spore germination was more sensitive to pressure around 300 MPa at 20 degrees C. Increasing processing temperatures during HPP enhanced the effect of sporulation medium pH (i.e., environmental pH) on the inactivation of B. cereus spores.