Combined effects of ultra-high hydrostatic pressure and mild heat on the inactivation of Bacillus subtilis

LWT - Food Science and Technology 68 (2016) 59e66

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Combined effects of ultra-high hydrostatic pressure and mild heat on the inactivation of Bacillus subtilis Jun Meng a, 1, Yi Gong a, 1, Ping Qian b, Jian-Yong Yu b, Xiao-Juan Zhang b, Rong-Rong Lu a, * a

State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, China b The Quartermaster Equipment Institute of the General Logistical Department of Chinese People's Liberation Army, 69 Lumicang, Beijing 10010, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 June 2015 Received in revised form 2 November 2015 Accepted 4 December 2015 Available online 11 December 2015

Effects of combined ultra-high hydrostatic pressure and mild heat (HPMH) treatments on the food-borne microorganism Bacillus subtilis were evaluated in this study. The B. subtilis culture (counted spores less than 2 log [CFU/mL]) was subjected to 100e500 MPa at 40
Ce60
C for 15 min at pH 7.0. Treatment with HPMH increased membrane permeability by 10%e89% as determined by the uptake of propidium iodide. Changes in membrane lipids, proteins and DNA were detected in regions 3000 to 2800 cm1 and 1300 to 900 cm1 by Fourier transformeinfrared spectroscopy (FT-IR). The membrane phospholipid molecules changed from a liquid crystalline state to a gel state, with a decrease in membrane fluidity. HPMH decreased the a-helix content（about 8e22%）while increased the random coil content (about 5e19%) of the cellular protein, which resulted in protein denaturation. Flow cytometry results indicated that HPMH treatment at 60
C caused 63% damage to the esterase activity of the cells more than HPMH treatment at 40
C. All of these results revealed the mechanism of HPMH, which is essential for the successful application of high hydrostatic pressure in neutral-pH food processing. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Ultra-high hydrostatic pressure Mild heat Combined inactivation Membrane permeability FT-IR spectroscopy

1. Introduction Thermal processing has been commonly applied to inactivate pathogenic and spoilage microorganisms to maintain food safety and increase shelf life, respectively. However, thermal processes can damage texture and alter the flavour and colour of food. Nonthermal methods can eliminate or minimize this degradation of food quality. Ultra-high hydrostatic pressure (UHHP) is a nonthermal technique that can maintain the natural qualities of food (Nguyen et al., 2010) as a result of its limited effects on covalent bonds (Yang, Jiang, Wang, Zhao, & Sun, 2009). However, in a neutral-pH environment, more than 600 MPa of pressure is required to eliminate pressureresistant bacteria (Chen, 2007a). Thus, hurdle technologies that make use of mild heat (Akhtar, Paredes-Sabja, Torres, & Sarker, ~o, 2013), high-intensity 2009; Zimmermann, Schaffner, & Araga ultrasound (Lee, Heinz, & Knorr, 2003) or carbon dioxide (Watanabe et al., 2005) have been used in combination with UHHP

* Corresponding author. E-mail address: [email protected] (R.-R. Lu). 1 These authors equally contributed to this work. http://dx.doi.org/10.1016/j.lwt.2015.12.010 0023-6438/© 2015 Elsevier Ltd. All rights reserved.

to inactivate the pathogens and spores in foods with a neutral pH. Bacteria are sensitive to temperature under pressurization. Studies have shown that some bacterial cells are resistant to UHHP at temperatures of 20
Ce35
C but become sensitive to pressurisation at higher temperatures (Alpas, Kalchayanand, Bozoglu, & Ray, 2000). Previous studies reported that the optimum process parameters for a 6-log-cycle reduction of Bacillus subtilis were 479 MPa at 46
C for 14 min (Gao & Jiang, 2005). However, under such process parameters the efficiencies of B. subtilis reduction in milk buffer and food matrix displayed some differences. Soybean protein, sucrose and pH significantly affected the reduction of B. subtilis (Gao, Ju, Qiu, & Jiang, 2007). In addition, the inactivation of bacterial cells (Alpas et al., 2000) and spores (Paredes-Sabja, Gonzalez, Sarker, & Torres, 2007) increased as the pH decreased under pressurisation. The composition of nutrients in the food system may affect the inactivation of microbial cells; for example, milk may exert a greater protective effect against inactivation by pressure than does water (Aouadhi et al., 2013). San Martín, Barbosa-C anovas & Swanson (2002) showed that even very high pressure levels (1000 MPa) at ambient temperature do not effectively inactivate bacterial spores. However, for some bacteria, effective inactivation can be achieved by combining UHHP

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J. Meng et al. / LWT - Food Science and Technology 68 (2016) 59e66

with temperatures greater than 40
C (Ju, Gao, Yao, & Qian, 2008; Kalchayanand, Dunne, Sikes, & Ray, 2004). Previous studies have concluded that a combination of pressure and moderate heat is always required to effectively inactivate spores of Bacillus species ry-Barraud, Gauberg, Masson, & Vidal, such as Bacillus anthracis (Cle 2004) and B. subtilis (Gao, Ju, & Jiang, 2006; Nguyen Thi Minh, Dantigny, Perrier-Cornet, & Gervais, 2010). B. subtilis is one of the most pressure-resistant Bacilli (Gao et al., 2006). Recently, the contamination of food products with B. subtilis has been shown to underlie food-borne diseases in humans (From, bal, & Granumh, 2005). Although the Pukall, Schumann, Hormaza primary target for UHHP in bacterial cells is believed to be the ~ as, & Mackey, 2010), the uncytoplasmic membrane (Klotz, Man derlying mechanism by which microorganisms are inactivated by UHHP and mild heat is still not fully understood. The objective of this study was to investigate the effects of ultrahigh hydrostatic pressure and mild heat (HPMH) in environments with a neutral pH by testing its effect on the food-borne microorganism B. subtilis in terms of membrane damage, the denaturation of protein and nucleic acid and metabolic activity. A full understanding of this combined mechanism is essential for the successful application of UHHP in the food-processing field.

2.3. Determination of viable counts Immediately after the HPMH treatment, the surviving bacteria were serially diluted in PBS and plated on tryptic soy agar to determine the viable cell counts. After aerobic incubation at 37
C for 48 h, the number of surviving cells was enumerated. Each count was calculated as the mean of three dishes for each dilution. The inactivation effect was expressed as Log (N0/N), where N0 was the initial count of the untreated sample and N was the corresponding viable number of cells after HPMH treatment. 2.4. Determination of substances that absorb ultraviolet light Absorption of ultraviolet light (UV) was carried out as described by Hong and Pyun (2001). After exposure to HPMH, the cells were centrifuged at 10,000 g for 10 min. The upper supernatant was removed, and the UV absorption was measured at a wavelength of 260 nm with a spectrophotometer (model UV-2450, Shimadzu, Japan). The measurements were made in triplicate and were corrected to account for absorbance by the medium used for HPMH treatment. 2.5. Determination of propidium iodide uptake by cells

2. Materials and methods 2.1. Bacterial strain and growth conditions B. subtilis ATCC 6633 was preserved in our laboratory and cultured in Oxoid's trypticase soy broth. The cultures were examined for the presence of spores by heating the cell suspension at 80
C for 10 min. The cell suspensions were diluted in ten-fold series in phosphate-buffered saline solution (PBS; pH 7.0). Selected dilutions of cell suspensions were plated on tryptic soy agar, following incubation at 37
C for 3 days, the number of the colonies were counted. The experiments were performed at least in triplicates. The concentration of the spores in all cultures used in the experiments was less than 102 CFU/ml (Shen, Urrutia Benet, Brul, & Knorr, 2005). The cells were harvested by centrifugation at 6,000 g at 4
C for 15 min, and the pellets were resuspended in 0.05 mol/L of phosphate-buffered saline solution (PBS; pH 7.0). The final concentration was 108e109 CFU/mL. The bacterial suspension was then stored at 4
C until the HPMH treatment within 1 h.

The uptake of propidium iodide (PI) by cells was performed according to the method of Klotz et al. (2010). To test whether the HPMH treatment caused membrane damage, the cells were stained with 3 mmol/L of a DNA-binding PI probe before treatment. Stock solutions of PI (Sigma) were prepared in sterile distilled water to a final concentration of 0.3 mmol/L. After exposure to HPMH as described above, the cells were incubated in the dark at 37
C for 10 min. The samples were then centrifuged and washed twice in 0.1 mol/L of PBS at pH 7.2. Fluorescence was measured by a spectrofluorophotometer (model F-7000, Hitachi, Japan) using an excitation wavelength of 495 nm and an emission wavelength of 615 nm. The fluorescence values obtained for the untreated cells were subtracted from the experimental values. This procedure was performed in triplicate. The fluorescence of cells stained with PI after heating at 90
C for 10 min was set as 100%, and the HPMH-treated cell membrane permeabilisation was expressed as the percentage of heat-treated cells. 2.6. Fourier transformeinfrared spectral measurements

2.2. HPMH processing Approximately 3 mL of bacterial suspension was placed in Corning tubes (Corning Incorporated, Corning, NY) and sealed after the removal of air bubbles. The samples were pressurised with a high-pressure unit (FPG5740, Stansted Fluid Power Co, UK) with 1,2-propanediol as a pressure-transmitting medium. The pressure level, time and temperature were computer-controlled. The temperature of the medium in the pressure vessel was measured by Ktype thermocouples during pressurisation. The vessel water jacket temperature was also controlled to obtain the desired temperature at the end of the process. After depressurisation, the samples were cooled in an ice bath and stored at 4
C for up to 6 h before enumeration. The cell cultures were pressurised at 100, 200, 300, 400 or 500 MPa for 15 min at 40
C, 50
C or 60
C. The time was fixed at 15 min because times exceeding this value have no industrial novas, Torres, & feasibility (Serment-Moreno, Fuentes, Barbosa-Ca Welti-Chanes, 2015). All HPMH experiments were performed in triplicate on separate days.

The Fourier transformeinfrared (FT-IR) measurement method of Al-Qadiri, Al-Alami, Al-Holy, and Rasco (2008) was used in this study with slight modifications. Untreated cells and HPMH-treated cells were washed twice and resuspended in sterile water. Suspension aliquots (200-mL) were transferred onto a ZnSe plate and dried at room temperature to produce a transparent film. The films were then directly analysed by FT-IR spectroscopy. All of the spectra were collected with a Nicolet Nexus 470 FT-IR spectrometer (Thermo Electron Corp, Waltham, MA). Attenuated total reflection (ATR) spectra were recorded from 4000 to 500 cm1 at a resolution of 4 cm1. Sixty-four interferograms were averaged for each spectrum. Omnic software (Thermo Electron Inc, San Jose, CA) was used to analyse the ATR spectra. The region of 1700 to 1600 cm1 in the FT-IR spectra mainly represented amide І, which indicated the secondary structure of proteins. A quantitative estimation of the secondary structure was made through second derivative calculation and Fourier selfdeconvolution curve-fitting analysis obtained with the use of Omnic software (Omnic 8.2, Thermo Electron Corp, Waltham, MA). Curve-fitting was conducted with Peakfit software(Peakfit 4.12,

J. Meng et al. / LWT - Food Science and Technology 68 (2016) 59e66

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SeaSolve Software Inc., Framingham, MA). The percentages of the a-helix, b-sheet, turns and random structures were determined using criteria described by Cai and Singh (1999). 2.7. Flow cytometric measurement Untreated, heat-treated and HPMH-treated cells were initially incubated for 10 min in the dark with 30 mmol/L of PI in an ice bath. Cells were then washed twice in PBS, and then 50 mmol/L of carboxyfluorescein diacetate (cFDA) (Molecular Probes Inc, Leiden, Netherlands) was added. Labelled cells were incubated at 37
C for 10 min to allow for the intracellular enzymatic conversion of cFDA into cF (Ananta, Heinz, & Knorr, 2004). Flow cytometric analysis was performed with a BD FACSCalibur flow cytometer (Becton, Dickinson and Company Inc, New Jersey, NJ). The flow rate was set at 400 events per second, up to a total of 10,000 events per sample. The data were displayed as dualparameter fluorescence density plots and analysed with CellQuest Pro software. Gate designation and possible cellular mechanisms involved as described by Ananta et al. (2004) are reported in Table 1. 2.8. Statistical analysis

Fig. 1. Inactivation of B. subtilis cells by 15-min HPMH treatments. The error bar represents standard deviation (n ¼ 3).

Table 2 Influence of HPMH on release of UV-absorbing substance from B. subtilis.

Results were expressed as mean values ± standard deviation of at least three independent experiments and analysed with a oneway analysis of variance. A P value below 0.05 was considered to be significant. SPSS 18.0 (SPSS Inc, Chicago, IL) for Windows was used in the statistical analysis. 3. Results 3.1. Influence of HPMH on the viability of B. subtilis The initial counts of B. subtilis before treatment were about 8e9 log (CFU/ml) (Fig. 1). Increasing the temperature from 40
C to 60
C under atmospheric pressure (0.1 MPa) had no effect on viability, but increasing the temperature from 40
C to 60
C under 500 MPa increased the reduction by 2.7 log (from 5.3 to 8.0 log [CFU/ml]). Treatment at 500 MPa and 40
C was not sufficiently severe to achieve 6-log reduction, but microbial inactivation of more than 6 log was achieved at 300e500 MPa and 60
C or at 500 MPa and 50
C.

Pressure (MPa) 0.1 100 200 300 400 500 Heat treatment

40
C 0.119 0.242 0.254 0.419 0.464 0.491 0.748

50
C ± ± ± ± ± ± ±

aA

0.03 0.03aA 0.04abA 0.02bcA 0.02cA 0.01cA 0.04e

60
C aA

0.106 ± 0.03 0.29 ± 0.01bA 0.408 ± 0.03bcAB 0.561 ± 0.03cdB 0.575 ± 0.01cdB 0.587 ± 0.01dB

0.179 0.323 0.485 0.578 0.671 0.703

± ± ± ± ± ±

0.01aA 0.02bA 0.01cB 0.02cdB 0.02deB 0.01eC

Triplicate measurements were obtained; the values are means ± standard deviations. Means in the same column labelled with different letters (aee) are significantly different (P < 0.05). Means in the same row labelled with different letters (AeC) are significantly different (P < 0.05).

no significant (P > 0.05) difference from that leaked from heattreated cells.

3.3. Influence of HPMH on the membrane permeability of B. subtilis 3.2. Influence of HPMH on release of UV-absorbing substance from B. subtilis During physical or chemical stress, cell components often leak out of the membrane, the cell components have been commonly measured by UV absorption to indicate the membrane damage (Hong & Pyun, 2001). The more severe treatment conditions resulted in the increased leakage of UV-absorbing substances in all treated samples (Table 2). Most HPMH-treated samples released fewer (P < 0.05) UV-absorbing substances than heat-treated samples, although the total amount of UV-absorbing substances released in the samples treated at 400e500 MPa and 60
C showed

PI could enter into membrane-compromised cells and bind to DNA, Klotz et al. (2010) has verified that high hydrostatic pressure could increase the membrane permeability of Escherichia coli using PI dye. The uptake of PI by HPMH-treated cells and heat-treated cells is shown in Fig. 2. The uptake of PI increased with pressure. Increasing temperature from 40
C to 60
C contributed a 19% enhancement (P < 0.05) in membrane permeability for cells treated at 100 MPa. The membrane permeability did not reach the level during heat treatment at 90
C for 10 min, although no detectable counts were observed after this heat treatment and HPMH treatment at 500 MPa and 60
C.

Table 1 The gate designation of the flow cytometric analysis. Gate

Fluorescence properties of cells

Possible explanation of the status of involved cellular mechanism

R1 R2 R3 R4

cFþ, PIþ cF-, PIþ cF-, PIcFþ, PI-

Active esterase membrane damaged Esterase activity not detectable membrane compromised Esterase not active or cF extruded out of the cells intact membrane Active esterase intact membrane

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membrane and cell wall components. Fig. 4B shows the nucleic acid denaturation associated with the antisymmetric (1220 cm1) and symmetric (~1080 cm1) P]O stretching mode of the phosphodiester backbone of nucleic acids. In addition, prominent changes were observed in the differences in CeOeC stretching vibrations at 1100 to 950 cm1, indicating that HPMH may also affect the structure of the peptidoglycan layer and lipopolysaccharide outer leaflet of the bacterial wall (Al-Qadiri, Al-Alami, et al., 2008). 3.5. Influence of HPMH on secondary structure of B. subtilis protein The relative contents of the secondary structure of B. subtilis cellular protein are shown in Table 3. It can be observed that the most common types of secondary structures of the cellular proteins in the untreated bacteria were a-helix and b-sheet. HPMH treatment decreased the content of a-helix but increased the contents of the other three structure types. Furthermore, the higher combined temperature shifted more a-helical structure to the three other types. Fig. 2. Cell membrane permeabilization of B. subtilis, as determined by PI uptake of HPMH treated cells. The fluorescence of cells stained with PI after heating at 90
C for 10 min was set as 100%. The error bar represents standard deviation (n ¼ 3). Identical letters indicate no significant differences (P < 0.05).

3.4. FT-IR spectra of HPMH-treated B. subtilis Changes in the cell components of B. subtilis after HPMH treatments were examined by FT-IR spectra (Fig. 3). The inactivation of the cells produced relatively similar spectra in two regions: 3000 to 2800 cm1 and 1300 to 900 cm1, both of which were dominated by bands assigned to the stretching vibration of amide І and nucleic acid. The spectra were converted to their second derivatives to facilitate discrimination between spectral features (Al-Qadiri, AlAlami, et al., 2008). Wave numbers of approximately 2855 cm1 and 2925 cm1 represent the symmetric and antisymmetric modes, respectively, of the eCH2 stretching vibrations of the membrane lipids. As Fig. 4A reveals, after HPMH treatment, the peak of band 2855 cm1 was shifted to 2858 cm1 and the peak of band 2925 cm1 was altered to 2929 cm1. Fig. 4B depicts the differences in the secondderivative spectra between HPMH-treated and control samples in the region from 1300 to 900 cm1. This region is characterised by vibrational features of cellular proteins, nucleic acids, cell

3.6. Inactivation of HPMH-treated cells as estimated by flow cytometric analysis Fresh bacteria showed a high degree of metabolic activity, and most of the cell population was seen in gate R4 (Fig. 5A). The cells treated by heat (90
C for 10 min) and labelled by PI were gated in R2, which suggested that the esterase activity in most of the heattreated cells was not detectable and that the membranes had been compromised (Fig. 5B). The cells treated solely by mild heat (40
C and 60
C) were mostly gated in R4, which indicated that mild heat had a limited influence on metabolic activity and that the cells were alive (Fig. 5C and D). Cells treated with HPMH at 500 MPa and 40
C retained residual esterase activity. Gate R1 held 82.2% of the dot plots, which indicated that the overall esterase activity was not significantly affected by HPMH at 500 MPa and 40
C (Fig. 5E). In contrast, HPMH treatment at 500 MPa and 60
C produced a significant change in esterase activity. Nearly 67% of the cells were distributed in gate R2 (Fig. 5F), which was similar to the distribution of cells seen after heat treatment. 4. Discussion Chen (2007b) reported that increasing pressure or temperature

Fig. 3. Representative FT-IR spectra of the control and the HPMH (500 MPa/60
C) treated samples of the B. subtilis strain.

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63

Fig. 4. Representative second-derivative FT-IR spectra of the control and the HPMH (500 MPa/60
C) treated samples of the B. subtilis strain. Part A shows regions of the membrane lipids (3000e2800 cm1). Part B shows regions of nucleic acids (1300e950 cm1).

alone Listeria monocytogenes reduction increased by less than 1 log, whereas increasing both bacterial reduction increased by about 4 log. The combined effect of pressure and mild heat was also observed in this study. UV-absorbing substances were detected in the upper supernatant of UHHP-treated samples. Leakage of UV-absorbing substances increased with pressure. Previous work by Klotz et al. (2010) also illustrated that PI is taken up, proteins leak, during pressure treatment. Interestingly, HPMH treatment at 500 MPa and 60
C almost completely inactivated the cells. However, the number of cells treated with HPMH at 500 MPa and 60
C that were stained by PI was significantly (P < 0.05) lower than that of cells that underwent heat treatment (90
C at 10 min). The observation that high pressure (HP)-treated cells were not

recoverable by plate counts but also were not stained with PI was interpreted as an indication of the presence of living, but metastamo, 1999). Ulmer, bolically inactive cells (Arroyo, Sanz, & Pre G€ anzle, and Vogel (2000) reported that although more than 99.99% of sublethally injured cells lost their ability to grow on media after HP treatment, PI staining was observed only after the cells had lost all metabolic activity, and 25%e50% membrane integrity was observed after HP treatment, resulting in a reduction in the viable cell counts of 4e6 orders of magnitude. Breeuwer and Abee (2000) also pointed out that microorganisms that had not formed colonies because they were dead, viable but non-culturable, injured, sublethally damaged, resting or inactive are not counted. The infrared ATR spectra of microbial cells under inactivation treatment reflect the composition of their cellular constituents,

Table 3 Influence of HPMH on secondary structure of B. subtilis protein. Temperature (
C)

Pressure (MPa)

a-helix (%)

b-sheet (%)

b-turn (%)

Random coil (%)

40

100 300 500 100 300 500

31.82 22.66 21.86 22.71 18.15 17.06 39.78 14.77

36.26 33.93 40.55 35.14 34.39 41.04 33.28 50.86

10.01 22.25 19.24 21.51 15.18 13.74 13.74 11.11

21.91 21.16 18.35 20.64 32.28 28.16 13.20 23.27

60

Untreated Heat treatment

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J. Meng et al. / LWT - Food Science and Technology 68 (2016) 59e66

Fig. 5. Flow cytometry dot plots of HPMH treated samples of B. subtilis strain. Part A shows the untreated sample. Part B shows the sample treated at 90
C/10 min. Part C shows the sample treated at 40
C. Part D shows the sample treated at 60
C. Part E shows the sample treated at 500 MPa/40
C. Part F shows the sample treated at 500 MPa/60
C.

J. Meng et al. / LWT - Food Science and Technology 68 (2016) 59e66

which includes water, fatty acids, proteins, polysaccharides and nucleic acids (Al-Qadiri, Al-Alami, et al., 2008; Al-Qadiri, Lin, AlHoly, Cavinato, & Rasco, 2008; Santivarangkna, Wenning, Foerst, & Kulozik, 2007). Previous studies have noted membrane phase transitions induced by UHHP in Lactobacillus plantarum. If the fatty acyl chains were ‘melted’ reaching a high conformational disorder, the peak shifted to a higher wave number. As a result, the physical state of the membranes of phospholipid molecules can change from a liquid crystalline state to a gel state with a decrease in membrane €nzle, & Winter, 2002). fluidity (Ulmer, Herberhold, Fashsel, Ga The membrane phase transition caused by HP is always reversible. Our FT-IR was completed as soon as possible after HPMH (within approximately 10 min). The differences between the control and HPMH-treated cells may occur during or before the reversible change. The results from 3000 to 2800 cm1 show that HPMH at 500 MPa and 60
C caused changes in the fatty acyl chains of the cell membrane. The shift of the wave numbers are conformation sensitive and thus respond to pressure- and temperatureinduced changes of the trans/gauche ratio in acyl chains. FT-IR spectroscopy in the range of 1700 to 1600 cm1 is a method to detect the secondary structure of proteins (Al-Qadiri, AlAlami, et al., 2008). In this study, it was concluded that the shifting of secondary structures from ordered to disordered states resulted in the decreased stability of proteins and further affected the metabolic process of the cells. Klotz et al. (2010) mentioned that the amount of protein released by the cells increased as the pressure rose to 400 MPa and then decreased because of protein aggrega~ as and Mackey (2004) also found that pressurisation tion. Man caused the condensation of proteins in both the exponential and stationary phases in E. coli. The FT-IR spectra of 1300 to 900 cm1 reflected the backbone ~ as change of the nucleic acid before and after HPMH treatment. Man and Mackey (2004) found that a condensation of nucleic acid occurred after treatment at 200 MPa for 8 min. Similar results were observed when E. coli was treated with a combination of UHHP and subzero temperatures (10 or 20
C) (Moussa, Perrier-Cornet, & Gervais, 2007). Many studies have shown that cells treated with high pressure retain residual esterase activity (Ananta et al., 2004; Ananta & Knorr, 2009; Shen, Bos, & Brul, 2009). Ananta and Knorr (2009) indicated that the esterase activity and the cell membrane of Lactobacillus rhamnosus ATCC were damaged at 75
C, but that cells exposed to 600 MPa still showed a high capacity for cF accumulation, even when a reduction of cells greater than 7 log was achieved. Our findings correspond with those of this research. HPMH treatment at 500 MPa and 40
C led to 5.3-log cell death, but more than 94% of the cells retained esterase activity (Fig 5E). With heat treatment at 90
C for 10 min, more than 83% of the cells were located in gate R2, which suggests that the thermal and HPMH treatments inactivated cells in different ways. It was interesting to note that the cells shifted from gate R1 to gate R2 when the processing temperature was elevated from 40
C to 60
C (Fig 5E and F). The increasing population in gate R2 indicated that the higher combination temperature assisted the UHHP treatment in reducing the energy-independent accumulation of cF, presumably by the inactivation of intracellular esterase to 10%. Increasing the temperature appeared to increase membrane permeabilisation, thus forcing esterase out of the cells. When separated from the cells, the esterase was much more easily damaged by the HPMH treatment. 5. Conclusions UHHP and mild heat displayed a combined effect in bacterial inactivation. As the pressure and temperature increased, the

65

proportion of bacteria with altered membrane permeability also increased. The FT-IR spectra reflected changes in the membrane lipids, proteins and nucleic acids caused by HPMH treatment. Flow cytometric analysis indicated that HPMH treatment at 500 MPa and 60
C can damage cellular esterase activity, leading to the death of B. subtilis. Our results increase the knowledge regarding the combination of UHHP and mild temperatures on the mechanisms of inactivation of B. subtilis cells and facilitate the application of UHHP in a hurdle system for the preservation of processed foods. Acknowledgements This study was supported by the grant from the National Natural Science Foundation of China (Nos. 31471696 & 31071491), “Twelfths 5-year” National Key Technology R&D Program of China (2012BAD33B05), the 863 project (2013AA102207), and the Priority Academic Program Development of Jiangsu Education Institutions. References Akhtar, S., Paredes-Sabja, D., Torres, J. A., & Sarker, M. R. (2009). Strategy to inactivate Clostridium perfringens spores in meat products. Food Microbiology, 26(3), 272e277. Al-Qadiri, H. M., Al-Alami, N. I., Al-Holy, M. A., & Rasco, B. A. (2008). Using fourier transform infrared (FT-IR) absorbance spectroscopy and multivariate analysis to study the effect of chlorine-induced bacterial injury in water. Journal of Agricultural and Food Chemistry, 56(19), 8992e8997. Al-Qadiri, H. M., Lin, M., Al-Holy, M. A., Cavinato, A. G., & Rasco, B. A. (2008). Detection of sublethal thermal injury in Salmonella enterica Serotype Typhimurium and Listeria monocytogenes using fourier transform infrared (FT-IR) spectroscopy (4000 to 600 cm1). Journal of Food Science, 73(2), 54e61. Alpas, H., Kalchayanand, N., Bozoglu, F., & Ray, B. (2000). Interactions of high hydrostatic pressure, pressurization temperature and pH on death and injury of pressure-resistant and pressure-sensitive strains of foodborne pathogens. International Journal of Food Microbiology, 60(1), 33e42. Ananta, E., Heinz, V., & Knorr, D. (2004). Assessment of high pressure induced damage on Lactobacillus rhamnosus GG by flow cytometry. Food Microbiology, 21(5), 567e577. Ananta, E., & Knorr, D. (2009). Comparison of inactivation pathways of thermal or high pressure inactivated Lactobacillus rhamnosus ATCC 53103 by flow cytometry analysis. Food Microbiology, 26(5), 542e546. vost, H., de Lamballerie, M., Maaroufi, A., & Mejri, S. Aouadhi, C., Simonin, H., Pre (2013). Inactivation of Bacillus sporothermodurans LTIS27 spores by high hydrostatic pressure and moderate heat studied by response surface methodology. LWT e Food Science and Technology, 50, 50e56. stamo, G. (1999). Response to high-pressure, lowArroyo, G., Sanz, P. D., & Pre temperature treatment in vegetables: determination of survival rates of microbial populations using flow cytometry and detection of peroxidase activity using confocal microscopy. Journal of Applied Microbiology, 86(3), 544e544. Breeuwer, P., & Abee, T. (2000). Assessment of viability of microorganisms employing fluorescence techniques. International Journal of Food Microbiology, 55, 193e200. Cai, S., & Singh, B. R. (1999). Identification of b-turn and random coil amide III infrared bands for secondary structure estimation of proteins. Biophysical Chemistry, 80(1), 7e20. Chen, H. (2007a). Use of linear, weibull, and log-logistic functions to model pressure inactivation of seven foodborne pathogens in milk. Food Microbiology, 24(3), 197e204. Chen, H. (2007b). Temperature-assisted pressure inactivation of Listeria monocytogenes in Turkey breast meat. International Journal of Food Microbiology, 117(1), 55e60. ry-Barraud, C., Gauberg, A., Masson, P., & Vidal, D. (2004). Combined effects of Cle high hydrostatic pressure and temperature for inactivation of Bacillus anthracis spores. Applied and Environmental Microbiology, 70(1), 635e637. From, C., Pukall, R., Schumann, P., Hormaz abal, V., & Granumh, P. E. (2005). Toxinproducing ability among Bacillus spp. outside the Bacillus cereus group. Applied and Environmental Microbiology, 71(3), 1178e1183. Gao, Y.-L., & Jiang, H.-H. (2005). Optimization of process conditions to inactivate Bacillus subtilis by high hydrostatic pressure and mild heat using response surface methodology. Biochemical Engineering Journal, 24(1), 43e48. Gao, Y.-L., Ju, X.-R., & Jiang, H.-H. (2006). Studies on inactivation of Bacillus subtilis spores by high hydrostatic pressure and heat using design of experiments. Journal of Food Engineering, 77(3), 672e679. Gao, Y.-L., Ju, X.-R., Qiu, W.-F., & Jiang, H.-H. (2007). Investigation of the effects of food constituents on Bacillus subtilis reduction during high pressure and moderate temperature. Food Control, 18(10), 1250e1257. Hong, S.-I., & Pyun, Y.-R. (2001). Membrane damage and enzyme inactivation of Lactobacillus plantarum by high pressure CO2 treatment. International Journal of Food Microbiology, 63(1e2), 19e28.

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