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Heat inactivation of Escherichia coli K12 MG1655: Effect of microbial metabolites and acids in spent medium
Journal of Thermal Biology 37 (2012) 72–78
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Heat inactivation of Escherichia coli K12 MG1655: Effect of microbial metabolites and acids in spent medium E.G. Velliou a, E. Van Derlinden a, A.M. Cappuyns a, A.H. Geeraerd b, F. Devlieghere c, J.F. Van Impe a,n a BioTeC—Chemical and Biochemical Process Technology and Control, Department of Chemical Engineering, Katholieke Universiteit Leuven, W. de Croylaan 46, B-3001 Leuven, Belgium b MEBIOS—Division of Mechatronics, Biostatistics and Sensors, Department of Biosystems (BIOSYST), Katholieke Universiteit Leuven, W. de Croylaan 42, B-3001 Leuven, Belgium c LFMFP—Laboratory of Food Microbiology and Food Preservation, Department of Food Technology and Nutrition, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 June 2011 Accepted 1 November 2011 Available online 20 November 2011
Aim: The effect of spent medium, obtained after different time–temperature pre-histories, on the heat inactivation of Escherichia coli K12 MG1655 is studied. Methods and results: Stationary E. coli cells were heated in BHI broth (initial pH 7.5) at different time– temperature scenarios, i.e., (1) 30 1C to 55 1C at 0.14 1C/min, (2) 30 1C to 42 1C at 0.14 1C/min and (3) 30 1C to 42 1C at 0.8 1C/min. After the heat treatment, spent medium was filter-sterilized, nonstressed cells were added and inactivation experiments took place at 54 1C and 58 1C. In all scenarios, increased resistance was observed. The main characteristics of the spent medium – compared to the unmodified BHI broth – are (1) the presence of proteins (proven via SDS-PAGE) and (2) a lower pH of approximately 6. Possibly, the increased resistance is due to these proteins and/or the lower pH. Further experiments revealed that each factor separately may lead to an increased heat resistance. Conclusions: It can be concluded that this increased heat resistance resulted from both the presence of the heat shock proteins in the spent medium and the lowered pH. Experiments, which separate both effects, showed that mainly the lower pH resulted in the increased thermotolerance. Significance and impact of study: This study may lead to a better understanding and control of the heat stress adaptation phenomenon as displayed by E. coli at lethal temperatures. Therefore, it contributes to an improved assessment of the effect of temperature during thermal processes in the food industry. & 2011 Published by Elsevier Ltd.
Keywords: Escherichia coli K12 MG1655 Heat resistance Heat shock proteins Heat stress Acid stress Cross protection
1. Introduction Recent food processing procedures tend towards less aggressive techniques like high hydrostatic pressure, irradiation and mild heat treatment. It is generally accepted that, in contrast to more traditional processes like pasteurization and sterilization, these new processing techniques maintain better textural and sensorial characteristics of fresh food products. However, as the applied conditions are less harsh, microbial survival might not be prevented and even stress adaptation can occur. Microorganisms reveal stress adaptation, i.e., the increase of resistance to environmental conditions that would normally be lethal (e.g., heat, acidity and chemical agents), by pre-exposure to a similar stress factor (Cebrian et al., 2009; Hassani et al., 2005,2006; Juneja and Marks, 2005; Valdramidis et al., 2006,2007) or a different kind of stress (Leyer and Johnson, 1993; Juneja and Novak, 2003; Skandamis et al., 2008,2009). The ability of a stress-adapted microorganism to
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resist when exposed to another kind of environmental stress is known as cross protection (Juneja and Novak, 2003). For instance, studies have proven that heat (or acid) shock may increase the resistance of bacteria to (heat), acids, ethanol and/or NaCl (see as examples Leyer and Johnson, 1993; Tetteth and Beuchat, 2003). When bacteria are exposed to an environmental stressing factor, they respond in several ways at the intracellular level. Most often, their response is the increased synthesis of specific proteins. When the stressing factor is high temperature, the produced shock proteins are known as Heat Shock Proteins (HSPs), e.g., GroEL, DnaK and DnaJ for Escherichia coli (Lindquist, 1986; Ohtsuka et al., 2007; Schlesinger, 1990; Yousef and Courtney, 2003). The basic mechanism of HSPs is to protect the cells from damages caused by heat via either a molecular chaperone function or a protease function. HSPs that act as molecular chaperones repair protein folding and therefore help the cell avoid denaturation. HSPs that are proteases degrade damaged proteins (Juneja et al., 1998; Juneja and Novak, 2003; Yura et al., 2000; Wick and Egli, 2004). HSPs are present not only in the cell but also at the extracellular level, acting as an alarm signal to cells (Ohtsuka et al., 2007; Wu and
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Tanguay, 2006). According to Rowburry (2001), extracellular components acting as stress sensors provide an earlier warning as compared to intracellular sensors. Usually, these components are proteins, which have as main function the switching on of the response to stress. After the detection of the stress by the Extracellular Sensing Components (ESCs) – produced in absence of the stress and activated by the stress – the production of Extracellular Induction Components (EICs) is switched on. Still according to this conceptual framework, EICs actually arise from ESCs (Rowburry and Goodson, 1999). These EICs remain present in the medium in absence of the stress. Since they are extracellular, they act as alarmones inducing tolerance to non-stressed cells or non-stressed regions leading to an intercellular communication, therefore allowing ‘cross-talk’ between cells, which are in different locations (Rowburry and Goodson, 1999; Rowburry, 2001,2003). In this paper, the effect of pre-exposure to mild heat stress on the thermotolerance of E. coli K12 is investigated. Specifically, the possible protective effect of extracellular components, i.e., as postulated in Rowburry and Goodson (1999) and Rowburry (2001,2003) is studied in more detail. Also, the role of acid (pre-) exposure, i.e., via cross protection, on the bacterial heat tolerance is considered.
2. Materials and methods 2.1. Inoculum preparation E. coli K12 MG1655 stock culture was stored at 80 1C in Brain Heart Infusion (BHI) broth (Oxoid Limited, Basingstoke, UK) with 25% (v/v) glycerol (Acros Organics, NJ, USA). For the preparation of the inoculum a loopful of the stock culture was transferred in 20 mL of BHI and was incubated at 37 1C on a rotary shaker (175 rpm) for 9.5 h. 20 mL of the cell suspension was transferred to 20 mL of fresh BHI and again incubated at 37 1C for 15 h. Early stationary phase cultures were harvested by centrifugation (1699 g, 2 min, 20 1C) and washing of portions of the cell suspensions in fresh BHI. 2.2. Preparation of the inoculated heating media Stationary phase E. coli K12 MG1655 cells were added in spent medium obtained after exposure to mild heat stress or in medium gained after acidification. The harvested cells prepared as described in paragraph 2.1 remained in each medium for approximately 30 min at room temperature. In a next step, stationary E. coli cells are inactivated at 54 1C and/or 58 1C in these spent media. A detailed description of the preparation and composition of each medium under study is given in the following paragraphs. 2.2.1. Pre-treatment of the medium (broth) for production of extracellular components Pre-treatment of the medium took place in sterile glass test tubes, in which 5 mL of cell suspension was pipetted. The tubes were immersed in a temperature controlled circulating water bath (GR150-S12, Grant Instruments Ltd, Shepreth, UK) where three different time–temperature scenarios were implemented. The scenarios were edited in a Labwises computer program and were (a) heating from 30 1C to 55 1C at 0.14 1C/min (Scenario 1, S1), (b) heating from 30 1C to 42 1C at 0.14 1C/min (Scenario 2, S2) and (c) heating from 30 1C to 42 1C at 0.8 1C/min (Scenario 3, S3). After the pre-treatment, the tubes were removed from the water bath, placed in an ice-water bath to cool down, and centrifuged (3220 g, 10 min, 20 1C). Supernatants were filtered with non-pyrogenic filters with 0.2 mm pores (SARSTEDT, Aktiengesellschaft & Co, Germany) in order to achieve microbial sterility. The pH of the
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spent media (Scenario 1, Scenario 2 and Scenario 3) was measured and was approximately 6, i.e., 5.9–6.1. The microbial population during the pre-treatment was monitored and it was observed that it remained stable at the level of 109 CFU/mL for all scenarios such that it can be assumed that cell lysis, i.e., the complete release of intracellular compounds into the spent medium, is limited. 2.2.2. pH readjustment of spent medium of Scenario 1 In order to separate the effect of the proteins from that of the lower pH, the pH level of Scenario 1 (produced as explained above) was readjusted to the level of approximately 7.5 (pH of unmodified BHI broth). The pH readjustment took place with addition of a specific volume of (sterile) 1 N KOH solution. 2.2.3. Ultra-filtration of spent medium of Scenario 1 Alternatively to the pH readjustment described above, spent medium of Scenario 1 was ultra-filtrated in order to remove the extracellular proteins. Ultra-filtration of the spent medium – produced as described above – took place in a (sterilized) Sterlitech HP4750 Stirred Cell device with the usage of membranes with pores of 4 kDa. This size of pores was chosen since it is much smaller compared to the proteins present in the medium of Scenario 1 (see Section 3.2). 2.2.4. Acidification of BHI The pH of unmodified BHI was approximately 7.5. Acidification took place with addition of 50% (v/v) acetic acid. The final pH of the acidified BHI was 6. 2.3. Thermal inactivation of E. coli at static temperatures Static inactivation experiments took place in sterile glass capillary tubes in which a volume of 60 mL cell suspension (prepared as described in paragraph 2.2) was pipetted. Tubes were then sealed by a gas flame and immersed in a water bath (GR150-S12, Grant Instruments Ltd, Shepreth, UK), at static temperatures of 54 1C and 58 1C. At regular times two capillaries were removed from the water bath, placed in an ice-water bath and analyzed within approximately 45 min. Decimal serial dilutions of the samples were prepared in a BHI solution and surface plated on BHI agar (1.2% (w/v)) using a Spiral Plater (Eddy Jet IUL Instruments, Barcelona, Spain). The volume plated was 49.2 mL. Plates were incubated for 24 h at 37 1C and colony forming units were enumerated. The detection limit was 3log CFU/mL. Each experiment was repeated in duplicate. 2.4. SDS-PAGE In order to investigate whether the components present in the pre-heated media were proteins, SDS-PAGE electrophoresis took place for all three pre-treated mediums (Scenario 1, 2 and 3). Fresh BHI was used as a control. 500 mL of the samples were mixed with 250 mL TCA (trichloro-acetic acid) 60% to a final concentration of 20% (v/v) and incubated on ice for 1 h. After the incubation the samples were centrifuged at 12,470 g for 15 min and the supernatants were removed. The pellets were washed with cold denatured ethanol (200 mL of ethanol per pellet), centrifuged at 12,470 g for 10 min and then, ethanol was removed and the protein pellets were dried in a heat block for 20 min. After drying, 40 mL of 2X loading buffer. (1.25 M Tris-HCl pH ¼ 6.8, SDS (Sodium Dodecyl Sulphate), 2-mercapto-ethanol, glycerol, chromophenol blue) was added to each pellet, and pellets were cooked for 5 min in water. 20 mL of each sample was loaded on to a SDS-PAA gel (12.5%), which
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was in the SDS-PAGE device. The proteins were separated by applying 300 mA and 300 V in the device. The loading buffer was Tris–glycine 10% (v/v) (glycine, Tris, SDS, pH78.3) and was allowed to run for approximately 1 h. Finally, the gels were stained with Commasie Staining Solution. 2.5. Data analysis The experimental data (cell density data) were log-transformed and plotted as a function of time. The inactivation model of Geeraerd et al. (2000) was fitted to the data (Eq. (1)): expðkmax SiÞ NðtÞ ¼ Nð0Þexpðkmax tÞ ð1Þ 1 þ ðexpðkmax SiÞ1Þexpðkmax tÞ with N [CFU/mL] the cell population, N(0) [CFU/mL] the initial cell population, kmax [1/min] the maximum specific inactivation rate, Si [min] the shoulder period and t [min] the time. The time for the fourth decimal reduction, t4D, was calculated, based on the estimated model parameters (Eq. (2)) (Buchanan et al., 1993): t 4D ¼ Si þ 4
lnð10Þ kmax
In a next step, stationary E. coli cells were inactivated at 54 1C and 58 1C in these spent media. Figs. 1 and 3 summarize the inactivation curves for the different experimental set-ups. Population inactivation was followed until approximately 3 log (CFU/mL) (i.e., the detection limit). For all cases, duplicate samples revealed similar behavior. All inactivation curves consist of a log-linear part, in most cases preceded by a so-called shoulder phase. This shoulder represents a period during which the bacterial cells are able to resynthesize a vital component and death ensues only when the rate of destruction exceeds the rate of synthesis. In the linear part, the rate of destruction is much higher than the rate of synthesis, and the microbial population decreases exponentially (Geeraerd et al., 2000). Generally, microbial heat inactivation dynamics, i.e., heat resistance, was determined by the duration of the shoulder length, Sl, and/or the inactivation rate denoted by kmax and/or an increase of the time for the fourth decimal reduction, t4D (Table 1). It should be mentioned that the values of the shoulder length Sl did not show a systematic trend as this parameter is – as with the lag phase for growth – not only determined by the actual inactivation conditions but also determined by (small differences in) the preceding conditions (Table 1).
ð2Þ
3.1. Effect of the time temperature pre-history on the spent medium
Modeling of the survival data was performed with GInaFiT (Version 1.5), a freeware add-in for Microsofts Excel (Geeraerd et al., 2005). Graphical illustrations were generated in MatLabs Version 7.4 (The Mathworks, Inc., Natick, USA). The experimental data were analyzed statistically via a t-student test in order to verify the significant indifference (Po0.05) of replicate experiments and the significant difference of the model parameters for different experimental conditions (P40.05).
When the bacteria were inactivated in spent medium resulting from S1 and S2, an increased heat resistance was observed compared to unmodified (fresh) Brain Heart Infusion broth (uBHI) (see Fig. 1). More specifically, an extension of the shoulder, a lower inactivation rate and an increase of the time for the fourth decimal reduction can be observed at 54 1C and 58 1C (see Table 1, Fig. 1(a) and (b), respectively). The extent of the increased resistance is approximately the same for S1 and S2, i.e., inactivation curves for the two scenarios run in parallel. For S3 (tested only at 54 1C), a reduction of the inactivation rate and a slight increase of the time for the fourth decimal reduction, compared to that of uBHI, were observed (Table 1, Fig. 1(a)). Also, a difference can be observed between inactivation in spent medium of S1/S2 and S3. The deviation between these scenarios (P4 0.05) is (partly) due to the fact that no shoulder phase can be observed for Scenario 3. As a result, inactivation starts earlier compared to S1 and S2. After approximately 50 min, the inactivation curves for S2 and S3 seem to evolve at the same rate. Two aspects of the spent medium that might explain the increased heat tolerance were further investigated: (1) the
3. Results In the present research, the possible protective effect of spent medium, obtained after exposure to mild heat stress, on the heat resistance of stationary phase (non pre-stressed) E. coli K12 MG1655 cells, was tested. Spent medium resulted from three heat treatments: (a) heating from 30 1C to 55 1C at 0.14 1C/min (Scenario 1, S1), (b) heating from 30 1C to 42 1C at 0.14 1C/min (Scenario 2, S2) and (c) heating from 30 1C to 42 1C at 0.8 1C/min (Scenario 3, S3).
Fig. 1. Microbial inactivation curves at (a) 54 1C and (b) 58 1C in (n) unmodified BHI broth, (o) Scenario 1, (þ ) Scenario 2 and (X) Scenario 3.
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Table 1 Values of the inactivation rate, kmax, and standard errors (SE) for all conditions under study. Inactivation medium
Exp.
kmax [1/min] SE 54 1C
Unmodified BHI (pH 7)
1 2
Scenario 1
1 2
Scenario 2
1 2
Scenario 3
1 2
Scenario 1 pH 7.5 (adjusted with KOH)
1 2
Ultrafiltrated Scenario 1
1 2
BHI of pH 6 (adjusted with acetic acid)
1 2
Sl [min] SE
t4D [min] SE
58 1C
54 1C
58 1C
1.26 10 1 5.76 10 3 1.28 10 1 5.60 10 3
1.79 100 1.13 10 2 1.13 100 1.27 10 1
22.5 4.7 21.3 4.9
4.5 0.2 3.1 0.7
93.0 5.8 95.3 5.7
9.6 0.3 11.2 1.2
7.79 10 2 3.62 10 3 7.81 10 2 2.20 10 3
1.07 100 2.52 10 2 1.08 100 3.30 10 2
36.5 8.7 43.6 4.4
6.7 1.1 7.0 0.3
154.6 10.3 161.5 5.5
15.4 0.3 15.5 0.4
6.82 10 2 2.27 10 3 7.34 10 2 2.01 10 3
1.39 100 5.74 10 2 1.53 100 5.00 10 2
No shoulder
No shoulder
8.4 0.2 8.8 0.2
134.9 4.5 125.5 3.5
15.0 0.4 14.8 0.3
–
No shoulder
–
–
No shoulder
–
106.7 4.8 103.3 5.1
2
8.63 10 3.89 10 3 8.92 10 2 4.37 10 3 2
– 1
54 1C
58 1C
–
9.82 10 3.98 10 3 9.31 10 2 5.59 10 3
8.07 10 1.97 10 2 8.38 10 1 1.82 10 2
18.9 6.1 13.9 8.2
2.8 0.8 2.9 0.3
112.8 7.2 113.0 10.1
14.2 0.5 13.9 0.4
5.68 10 2 4.03 10 3 5.67 10 2 2.98 10 3
–
40.6 13.9 36.7 10.5
–
–
–
202.7 18.1 199.1 13.6
–
34.5 3.3 25.6 6.8
7.0 0.3 4.6 0.5
152.7 8.2 143.8 4.1
14.7 0.8 15.3 0.6
2
8.43 10 1.82 10 3 7.25 10 2 2.58 10 3
presence of (extracellular) proteins (Figs. 2 and 3), and (2) the reduced pH of the spent medium (approximately 6 compared to 7.5 for uBHI) (Fig. 3). 3.2. Effect of the (extracellular) proteins 3.2.1. Presence of (extracellular) proteins SDS-PAGE analysis of the three pre-treated media indicated the presence of a variety of proteins compared to the proteins present in uBHI (Fig. 2). In the case of S1 and S2, a protein band with a molecular weight of approximately 100 kDa was present (more pronounced for S2). Furthermore, protein bands with a molecular weight of 60 kDa, approximately 50 kDa (more pronounced for S2), and 37 kDa were observed. For S3, only a protein band of approximately 60 kDa was observed. However, this band is not as pronounced as it is for S1 and S2.
– 1
6.65 10 1.27 10 2 6.42 10 1 2.12 10 2
100 kDa 75 kDa 50 kDa 37 kDa
25 kDa 20 kDa C1
3.2.2. pH readjustment of spent medium In order to distinguish between the protein effect and the pH effect, stationary phase E. coli cells were added in spent medium of S1 with the pH readjusted to 7.5 (pH of uBHI) and inactivated at 54 1C and 58 1C. After pH adaptation, the stationary phase cells remain more heat resistant than cells inactivated in uBHI (Table 1, Fig. 3, P40.05). The increase in the bacterial tolerance is more pronounced at 58 1C. 3.2.3. Ultra-filtration of spent medium The protein effect on the bacterial heat resistance was studied in more detail by inactivating the stationary phase cells in ultrafiltrated S1 spent medium at 54 1C. The pH of the spent medium
S1
S2
S3
uBHI
S1(UF)
Fig. 2. Image analysis of the SDS-PAGE gel: (C1) the letter of the gel, (S1) Scenario 1, (S2) Scenario 2, (S3) Scenario 3, (uBHI) unmodified BHI broth and (S1þ (UF)) Scenario 1 after ultrafiltrarion.
remained equal to 6 after the ultra-filtration. The ultra-filtrated medium was analyzed via SDS-PAGE in order to check whether all the proteins were removed. As can be seen on the gel of Fig. 2, no protein bands were present in the ultra-filtrated spent medium of S1, i.e., the only remaining stressing factor was the low pH. Heat inactivation at 54 1C in spent BHI with and without the extracellular proteins was rather similar, i.e., removing the proteins did not significantly change the thermotolerance of E. coli (Table 1, Fig. 3(a)).
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Fig. 3. Microbial inactivation curves at (a) 54 1C and (b) 58 1C in (n) unmodified BHI broth, (o) Scenario 1, (þ) Scenario 1 at pH 7.5 (adapted with KOH), (X) Scenario 1 after ultrafiltration and (B) BHI of pH 6 (adapted with acetic acid).
3.3. Effect of acidity of the medium In a second step, the effect of low pH on the heat resistance of E. coli was studied. Heat inactivation experiments were performed in uBHI of pH 6 at 54 1C and 58 1C. The pH of the broth was adjusted with addition of 50% (v/v) acetic acid. Acetic acid was chosen for the acidification of the BHI as it is the main acid produced by E. coli during growth in a glucose rich environment (such as BHI). As can be seen in Table 1 and Fig. 3, increased heat resistance was observed when the bacteria were inactivated in acidified BHI, compared to the heat inactivation in unmodified BHI at pH 7.5 (P40.05). More specifically, at 54 1C heat resistance was increased, i.e., an extended shoulder, a reduced inactivation rate and an increase of the t4D, can be observed. Generally, the thermotolerance was approximately the same as observed when the bacterial cells were inactivated in spent medium of S1 (Table 1, Fig. 3 (a)). At 58 1C, the increased heat tolerance was even higher for the acidified uBHI, compared to the spent medium of S1, i.e., the inactivation rate is lower (Table 1). The duration of the shoulder and the t4D were approximately the same both for S1 and uBHI at pH 6 (Table 1, Fig. 3 (b)). These results seem to suggest that the pH effect dominates the extracellular protein effect.
4. Discussion The inactivation of stationary E. coli cells in spent medium, obtained after exposure to a mild heat stress, occured slower compared to fresh Brain Heart Infusion medium. Two aspect of the spent medium can be brought forward as an explanation for the increased heat resistance: (1) the presence of specific proteins in the spent medium, and (2) the slightly reduced pH of the spent medium. The spent medium was produced by growing E. coli under three different dynamic temperature conditions (S1, S2 and S3). For all three cases, the final temperature was situated in the superoptimal temperature region for E. coli, which triggered the heat shock response and, therefore, the production of specific (heat shock) proteins, which increase cell stability at higher temperatures. The inactivation in spent medium of S1 and S2
was almost identical, which suggests that the final temperature has no influence on the heat resistance. It is, however, most likely necessary that superoptimal temperatures (T437 1C) are reached such that the heat shock response is induced. Fig. 2 shows that the same proteins were present in the spent medium of S1 and S2 and that concentrations were in the same range, i.e., the intensity of the protein bands is similar. A small difference can be observed between inactivation curves of S1 and S2 – with heating rate 0.14 1C/min – and S3 – with heating rate 0.8 1C/min. More specifically, for S2 the inactivation rate was smaller and the shoulder phase and the time for the fourth decimal reduction longer, compared to S3 (Table 1, Fig. 3(a)). As the initial and final temperatures of these scenarios were identical (initial temperature: 30 1C, final temperature: 42 1C), this observation seems to suggest that the difference in the heating rate (S2: 0.14 1C/min, S3: 0.8 1C/min) results in different heat responses, which lead to different inactivation dynamics. A study of Tsuchido et al. (1982) showed that the slower E. coli K12 cells were heated from 0 1C to 50 1C, the higher the heat resistance of these cells was at 50 1C. Valdramidis et al. (2006, 2007) also indicated that, for a temperature up-shift from 30 1C to 55 1C, lower heating rates resulted in higher heat resistance of E. coli. Most likely, cells are more able to adjust to the environment and become more heat resistant when a slower temperature increase is imposed (Mackey and Derrick, 1987; Juneja and Marks, 2005). It is possible that in S2, i.e., with a slower heating rate (0.14 1C/min), the cells have more time to adapt to the rising temperature and, therefore produce more components or higher concentrations, compared to S3, which possibly results in an increased resistance of non pre-stressed cells. This hypothesis is confirmed by the SDS-page gel shown in Fig. 2: less or less pronounced protein bands are observed for S3, compared to S1 and S2. The molecular weights of some of the proteins found in the spent medium of S1 (Fig. 3) were similar to those of the most known HSPs (Ohtsuka et al., 2007; Rowburry, 2003; Juneja et al., 1998). Specifically the proteins of 100 kDa had similar molecular weight to the CIpA, ClpB, CIpC, CIpX and CIpY proteins of the HSP100 family, which contribute to the induction of thermotolerance (Ohtsuka et al., 2007). Generally, similar proteins can be recovered for S1 and S2, which resulted in almost identical
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inactivation kinetics, in contrast to S3 where less proteins were observed and inactivation occurred faster. This observation suggests that the presence of specific proteins somehow alerts or protects the non pre-stressed cells leading to an increased thermotolerance. These findings are in agreement with the conclusion of Heredia et al. (2009), who studied the involvement of extracellular compounds in the protection of Clostridium perfringens to heat, and showed that extracellular factors (proteins) produced by heat shocked cells have a thermoprotective function for non-stressed cells. Next to the presence of the proteins, the biggest difference – relevant for microbial inactivation kinetics – between fresh BHI and the spent medium was the pH. During the heat treatments, E. coli produced acetic acid, which resulted in a lower medium pH, i.e., 6, compared to approximately 7.5 of fresh BHI. This acidified environment was an additional stressing factor that may affect the (heat) resistance of E. coli. A series of inactivation experiments at 54 and 58 1C showed that the pH has a significant effect on the heat inactivation of E. coli. A first experiment, in which the pH of the spent medium (S1) was adapted to 7.5 (pH of uBHI), showed that the heat resistance was higher when compared to the uBHI, i.e., a slight prolongation of the shoulder (Table 1), a reduction of the inactivation rate and an increase of the t4D (Table 1) can be observed for both studied temperatures. The increase on the heat tolerance was more pronounced at 58 1C (Fig. 3). This finding is an indication that the presence of the proteins somehow increases the thermotolerance. In a second experiment, stationary phase cells were inactivated at 54 1C in ultra-filtrated spent medium (S1) at its original pH ( E6). By ultrafiltatrion, all the proteins present in the spent medium were efficiently removed (Fig. 2). The bacterial heat resistance did not alter after removing the proteins, i.e., inactivation dynamics were identical to the complete S1 spent medium. The observation that the thermotolerance remained at the same level in absence of the proteins is an indication that, most likely, the mainly low pH of the spent medium triggers the cells leading to an increased heat resistance. In a third series of experiments, the beneficial effect of the lower pH on the microbial heat resistance was confirmed. Stationary E. coli cells were inactivated in fresh BHI of pH 6 at 54 1C and 58 1C. The pH of the broth was readjusted with addition of 50% (v/v) acetic acid to mimic a real pH decrease as a result of microbial growth. Generally, increased resistance can be observed for the acidified fresh BHI compared to the uBHI (pH 7.5) and the S1 spent medium at pH 6 (Table 1, Fig. 3). Most likely, the different composition of the spent medium, i.e., less nutritional components, other (toxic) metabolic products and a different amount of acid, i.e., different pH level of the growth medium, resulted in a different microbial response to the stressful conditions. The increased heat resistance observed in the acidified fresh BHI corresponds with findings of other studies. Velliou et al. (2011) have shown that the heat resistance of E. coli at 54 1C and 58 1C increases when (pre-)exposed to different kinds of acids, i.e., acetic, lactic and hydrochloric acid. The extent of the resistance is dependent on the type of acid and on the quantity added. Acidification of the growth medium with acetic acid at a pH level of 6 and 5.5 shortly before heat inactivation led to an increased bacterial heat tolerance at 54 1C and 58 1C. Tetteth and Beuchat (2003) have proven that a heat and/or an acid shock can lead to an increase of the concentration of heat shock proteins, and finally to an increased acid tolerance of Shigella flexneri. According to Jorgensen et al. (1999), the presence of lactic acid has a specific positive effect on the response of Listeria monoytogenes to heat. Skandamis et al. (2008) observed that there is increased heat resistances of stationary phase cultures of L. monocytogenes at 52, 57 and 63 1C, following exposure to an acidic environment (acidified with lactic acid).
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5. General conclusions When stationary phase E. coli cells were heated in BHI under dynamic temperature conditions that reached the superoptimal temperature range, pH lowered due to acid production and heat shock proteins were produced as a response to the mild temperature stress. When unstressed stationary E. coli cells were inactivated in this spent medium, an increase in thermotolerance was observed. Based on a series of inactivation experiments, it can be concluded that this increased heat resistance resulted from both the presence of the heat shock proteins in the spent medium and the lowered pH. Experiments, which separate both effects, showed that mainly the lower pH resulted in the increased thermotolerance. Most likely, the presence of acetic acid induced an acid stress response, which resulted in a cross protection for the heat. This knowledge is of great importance in the food processing industry and could contribute in a more efficient design of emerging technologies and processes used in our days, in which milder heating conditions are applied compared to the conventional methods for achieving pasteurization.
Acknowledgments We would like to thank Prof. Jef Anne´ and Philip Gutschoven (Department of Microbiology and Immunology, Rega Institute, Katholieke Universiteit Leuven) for providing equipment and their help in the SDS-PAGE electrophoresis and Prof. Bart Van der Bruggen for kindly providing the ultrafiltration device in his laboratory. This work was supported by projects PFV/10/002 (Center of Excellence-OPTEC Optimization in Engineering) of the Research Council of the Katholieke Universiteit Leuven, project KP/09/005 (SCORES4CHEM) of the Industrial Research Fund and the Belgian Program on Interuniversity Poles of Attraction, initiated by the Belgian Federal Science Policy Office. E. Van Derlinden is supported by the postdoctoral grant PDMK/10/122 of the K.U.Leuven Research Fund. J. Van Impe holds the chair Safety Engineering sponsored by the Belgian chemistry and life sciences federation essenscia.
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Juneja, V.K., Novak, J.S., 2003. Adaptation of foodborne pathogens to stress from exposure to physical intervention strategies. In: Yousef, A.E., Juneja, V.K. (Eds.), Microbial Stress Adaptation and Food Safety. CRC Press, Boca Raton, pp. 159–211. Juneja, V.K., Marks, H.M., 2005. Heat resistance kinetics variation among various isolates of Escherichia Coli. Innov. Food Sci. Emerg. Technol. 6, 155–161. Leyer, G.J., Johnson, E.A., 1993. Acid adaptation induces cross-protection against environmental stresses in Salmonella typhimurium. Appl. Environ. Microbiol. 59, 1842–1847. Lindquist, S., 1986. The heat-shock response. Annu. Rev. Biochem. 55, 1151–1191. Mackey, B.M., Derrick, C.M., 1987. Changes in the heat resistance of Salmonella typhimurium during heating at rising temperatures. Lett. Appl. Microbiol. 4, 13–16. Ohtsuka, K., Kawashima, D., Asai, M., 2007. Dual functions of heat shock proteins: molecular chaperones inside of cells and danger signals outside of cells. Therm. Med. 23, 11–22. Rowburry, R.J., Goodson, M., 1999. An extracellular stress-sensing protein is activated by heat and u.v. irradiation as well as by mild acidity, the activation producing an acid tolerance-inducing protein. Lett. Appl. Microbiol. 29, 10–14. Rowburry, R.J., 2001. Cross-talk involving extracellular sensors and extracellular alarmones gives early warning to unstressed Escherichia coli of impending lethal chemical stress and leads to induction of tolerance responses. J. Appl. Microbiol. 90, 677–696. Rowburry, R.J., 2003. Physiology and molecular basis of stress adaptation, with particular reference to the subversion of stress adaptation and to the involvement of extracellular components in adaptation. In: Yousef, A.E., Juneja, V.K. (Eds.), Microbial Stress Adaptation and Food Safety. , CRC Press, Boca Raton, pp. 159–211. Schlesinger, M.J., 1990. Heat shock proteins. J. Biol. Chem. 265, 12111–12114. Skandamis, P.N., Stopforth, J.D., Yoon, Y., Kendall, P.A., Sofos, J.N., 2009. Heat and acid tolerance responses of Listeria monocytogenes as affected by sequential exposure to hurdles during growth. J. Food Prot. 72, 1412–1418.
Skandamis, P.N., Yoon, Y., Stopforth, D., Kendall, P.A., Sofos, J.N., 2008. Heat and acid tolerance of Listeria monocytogenes after exposure to single and multiple sublethal stresses. Food Microbiol. 25, 294–303. Tetteth, G.L., Beuchat, R., 2003. Exposure of Shigella flexneri to acid stress and heat shock enhances acid tolerance. Food Microbiol. 20, 179–185. Tsuchido, T., Hayashi, M., Takano, M., Shibasaki, I., 1982. Alternation of thermal resistance of microorganisms in a non-isothermal heating process. J. Antibact. Antifung. Agents 10, 105–109. Valdramidis, V.P., Geeraerd, A.H., Bernaerts, K., Van Impe, J.F., 2006. Microbial dynamics versus mathematical model dynamics: the case of microbial heat resistance induction. Innov. Food Sci. Emerg. Technol. 7, 118–125. Valdramidis, V.P., Geeraerd, A.H., Van Impe, J.F., 2007. Stress adaptive responses by heat under the microscope of predictive microbiology. J. Appl. Microbiol. 103, 1922–1930. Velliou, E.G., Van Derlinden, E., Cappuyns, A.M., Nikolaidou, E., Geeraerd, A.H., Devlieghere, F., Van Impe, J.F., 2011. Towards the quantification of the effect of acid treatment on the heat tolerance of Escherichia coli K12 at lethal temperatures. Food. Microbiol. 28, 702–711. Wu, T., Tanguay, R.M., 2006. Antibodies against heat shock proteins in environmental stresses and diseases: friend or foe? Cell Stress Chap. 11, 1–12. Wick, L.M., Egli, T., 2004. Molecular components on physiological stress response in Escherichia coli. In: Enfors, S.O. (Ed.), Physiological Stress Responses in Bioprocesses, Advances in Biochemical Engineering/Biotechnology. vol. 89, pp. 1–46. Yousef, A.E., Courtney, P.D., 2003. Basics of stress adaptation and implications in new-generation foods. In: Yousef, A.E., Juneja, V.K. (Eds.), Microbial Stress Adaptation and Food Safety. , CRC Press, Boca Raton, pp. 1–32. Yura, T., Kanemori, M., Morita, T., 2000. The heat shock response: regulation and function. In: Storz, G., Aronis, R.H. (Eds.), Bacterial Stress Responses. ASM Press, Washington, DC, pp. 3–18In: Storz, G., Aronis, R.H. (Eds.), Bacterial Stress Responses. , ASM Press, Washington, DC, pp. 3–18 (20036-2804).
Contents lists available at SciVerse ScienceDirect
Journal of Thermal Biology journal homepage: www.elsevier.com/locate/jtherbio
Heat inactivation of Escherichia coli K12 MG1655: Effect of microbial metabolites and acids in spent medium E.G. Velliou a, E. Van Derlinden a, A.M. Cappuyns a, A.H. Geeraerd b, F. Devlieghere c, J.F. Van Impe a,n a BioTeC—Chemical and Biochemical Process Technology and Control, Department of Chemical Engineering, Katholieke Universiteit Leuven, W. de Croylaan 46, B-3001 Leuven, Belgium b MEBIOS—Division of Mechatronics, Biostatistics and Sensors, Department of Biosystems (BIOSYST), Katholieke Universiteit Leuven, W. de Croylaan 42, B-3001 Leuven, Belgium c LFMFP—Laboratory of Food Microbiology and Food Preservation, Department of Food Technology and Nutrition, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 June 2011 Accepted 1 November 2011 Available online 20 November 2011
Aim: The effect of spent medium, obtained after different time–temperature pre-histories, on the heat inactivation of Escherichia coli K12 MG1655 is studied. Methods and results: Stationary E. coli cells were heated in BHI broth (initial pH 7.5) at different time– temperature scenarios, i.e., (1) 30 1C to 55 1C at 0.14 1C/min, (2) 30 1C to 42 1C at 0.14 1C/min and (3) 30 1C to 42 1C at 0.8 1C/min. After the heat treatment, spent medium was filter-sterilized, nonstressed cells were added and inactivation experiments took place at 54 1C and 58 1C. In all scenarios, increased resistance was observed. The main characteristics of the spent medium – compared to the unmodified BHI broth – are (1) the presence of proteins (proven via SDS-PAGE) and (2) a lower pH of approximately 6. Possibly, the increased resistance is due to these proteins and/or the lower pH. Further experiments revealed that each factor separately may lead to an increased heat resistance. Conclusions: It can be concluded that this increased heat resistance resulted from both the presence of the heat shock proteins in the spent medium and the lowered pH. Experiments, which separate both effects, showed that mainly the lower pH resulted in the increased thermotolerance. Significance and impact of study: This study may lead to a better understanding and control of the heat stress adaptation phenomenon as displayed by E. coli at lethal temperatures. Therefore, it contributes to an improved assessment of the effect of temperature during thermal processes in the food industry. & 2011 Published by Elsevier Ltd.
Keywords: Escherichia coli K12 MG1655 Heat resistance Heat shock proteins Heat stress Acid stress Cross protection
1. Introduction Recent food processing procedures tend towards less aggressive techniques like high hydrostatic pressure, irradiation and mild heat treatment. It is generally accepted that, in contrast to more traditional processes like pasteurization and sterilization, these new processing techniques maintain better textural and sensorial characteristics of fresh food products. However, as the applied conditions are less harsh, microbial survival might not be prevented and even stress adaptation can occur. Microorganisms reveal stress adaptation, i.e., the increase of resistance to environmental conditions that would normally be lethal (e.g., heat, acidity and chemical agents), by pre-exposure to a similar stress factor (Cebrian et al., 2009; Hassani et al., 2005,2006; Juneja and Marks, 2005; Valdramidis et al., 2006,2007) or a different kind of stress (Leyer and Johnson, 1993; Juneja and Novak, 2003; Skandamis et al., 2008,2009). The ability of a stress-adapted microorganism to
n
Corresponding author. Fax: þ32 16 32 2991. E-mail address: [email protected] (J.F. Van Impe).
0306-4565/$ - see front matter & 2011 Published by Elsevier Ltd. doi:10.1016/j.jtherbio.2011.11.001
resist when exposed to another kind of environmental stress is known as cross protection (Juneja and Novak, 2003). For instance, studies have proven that heat (or acid) shock may increase the resistance of bacteria to (heat), acids, ethanol and/or NaCl (see as examples Leyer and Johnson, 1993; Tetteth and Beuchat, 2003). When bacteria are exposed to an environmental stressing factor, they respond in several ways at the intracellular level. Most often, their response is the increased synthesis of specific proteins. When the stressing factor is high temperature, the produced shock proteins are known as Heat Shock Proteins (HSPs), e.g., GroEL, DnaK and DnaJ for Escherichia coli (Lindquist, 1986; Ohtsuka et al., 2007; Schlesinger, 1990; Yousef and Courtney, 2003). The basic mechanism of HSPs is to protect the cells from damages caused by heat via either a molecular chaperone function or a protease function. HSPs that act as molecular chaperones repair protein folding and therefore help the cell avoid denaturation. HSPs that are proteases degrade damaged proteins (Juneja et al., 1998; Juneja and Novak, 2003; Yura et al., 2000; Wick and Egli, 2004). HSPs are present not only in the cell but also at the extracellular level, acting as an alarm signal to cells (Ohtsuka et al., 2007; Wu and
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Tanguay, 2006). According to Rowburry (2001), extracellular components acting as stress sensors provide an earlier warning as compared to intracellular sensors. Usually, these components are proteins, which have as main function the switching on of the response to stress. After the detection of the stress by the Extracellular Sensing Components (ESCs) – produced in absence of the stress and activated by the stress – the production of Extracellular Induction Components (EICs) is switched on. Still according to this conceptual framework, EICs actually arise from ESCs (Rowburry and Goodson, 1999). These EICs remain present in the medium in absence of the stress. Since they are extracellular, they act as alarmones inducing tolerance to non-stressed cells or non-stressed regions leading to an intercellular communication, therefore allowing ‘cross-talk’ between cells, which are in different locations (Rowburry and Goodson, 1999; Rowburry, 2001,2003). In this paper, the effect of pre-exposure to mild heat stress on the thermotolerance of E. coli K12 is investigated. Specifically, the possible protective effect of extracellular components, i.e., as postulated in Rowburry and Goodson (1999) and Rowburry (2001,2003) is studied in more detail. Also, the role of acid (pre-) exposure, i.e., via cross protection, on the bacterial heat tolerance is considered.
2. Materials and methods 2.1. Inoculum preparation E. coli K12 MG1655 stock culture was stored at 80 1C in Brain Heart Infusion (BHI) broth (Oxoid Limited, Basingstoke, UK) with 25% (v/v) glycerol (Acros Organics, NJ, USA). For the preparation of the inoculum a loopful of the stock culture was transferred in 20 mL of BHI and was incubated at 37 1C on a rotary shaker (175 rpm) for 9.5 h. 20 mL of the cell suspension was transferred to 20 mL of fresh BHI and again incubated at 37 1C for 15 h. Early stationary phase cultures were harvested by centrifugation (1699 g, 2 min, 20 1C) and washing of portions of the cell suspensions in fresh BHI. 2.2. Preparation of the inoculated heating media Stationary phase E. coli K12 MG1655 cells were added in spent medium obtained after exposure to mild heat stress or in medium gained after acidification. The harvested cells prepared as described in paragraph 2.1 remained in each medium for approximately 30 min at room temperature. In a next step, stationary E. coli cells are inactivated at 54 1C and/or 58 1C in these spent media. A detailed description of the preparation and composition of each medium under study is given in the following paragraphs. 2.2.1. Pre-treatment of the medium (broth) for production of extracellular components Pre-treatment of the medium took place in sterile glass test tubes, in which 5 mL of cell suspension was pipetted. The tubes were immersed in a temperature controlled circulating water bath (GR150-S12, Grant Instruments Ltd, Shepreth, UK) where three different time–temperature scenarios were implemented. The scenarios were edited in a Labwises computer program and were (a) heating from 30 1C to 55 1C at 0.14 1C/min (Scenario 1, S1), (b) heating from 30 1C to 42 1C at 0.14 1C/min (Scenario 2, S2) and (c) heating from 30 1C to 42 1C at 0.8 1C/min (Scenario 3, S3). After the pre-treatment, the tubes were removed from the water bath, placed in an ice-water bath to cool down, and centrifuged (3220 g, 10 min, 20 1C). Supernatants were filtered with non-pyrogenic filters with 0.2 mm pores (SARSTEDT, Aktiengesellschaft & Co, Germany) in order to achieve microbial sterility. The pH of the
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spent media (Scenario 1, Scenario 2 and Scenario 3) was measured and was approximately 6, i.e., 5.9–6.1. The microbial population during the pre-treatment was monitored and it was observed that it remained stable at the level of 109 CFU/mL for all scenarios such that it can be assumed that cell lysis, i.e., the complete release of intracellular compounds into the spent medium, is limited. 2.2.2. pH readjustment of spent medium of Scenario 1 In order to separate the effect of the proteins from that of the lower pH, the pH level of Scenario 1 (produced as explained above) was readjusted to the level of approximately 7.5 (pH of unmodified BHI broth). The pH readjustment took place with addition of a specific volume of (sterile) 1 N KOH solution. 2.2.3. Ultra-filtration of spent medium of Scenario 1 Alternatively to the pH readjustment described above, spent medium of Scenario 1 was ultra-filtrated in order to remove the extracellular proteins. Ultra-filtration of the spent medium – produced as described above – took place in a (sterilized) Sterlitech HP4750 Stirred Cell device with the usage of membranes with pores of 4 kDa. This size of pores was chosen since it is much smaller compared to the proteins present in the medium of Scenario 1 (see Section 3.2). 2.2.4. Acidification of BHI The pH of unmodified BHI was approximately 7.5. Acidification took place with addition of 50% (v/v) acetic acid. The final pH of the acidified BHI was 6. 2.3. Thermal inactivation of E. coli at static temperatures Static inactivation experiments took place in sterile glass capillary tubes in which a volume of 60 mL cell suspension (prepared as described in paragraph 2.2) was pipetted. Tubes were then sealed by a gas flame and immersed in a water bath (GR150-S12, Grant Instruments Ltd, Shepreth, UK), at static temperatures of 54 1C and 58 1C. At regular times two capillaries were removed from the water bath, placed in an ice-water bath and analyzed within approximately 45 min. Decimal serial dilutions of the samples were prepared in a BHI solution and surface plated on BHI agar (1.2% (w/v)) using a Spiral Plater (Eddy Jet IUL Instruments, Barcelona, Spain). The volume plated was 49.2 mL. Plates were incubated for 24 h at 37 1C and colony forming units were enumerated. The detection limit was 3log CFU/mL. Each experiment was repeated in duplicate. 2.4. SDS-PAGE In order to investigate whether the components present in the pre-heated media were proteins, SDS-PAGE electrophoresis took place for all three pre-treated mediums (Scenario 1, 2 and 3). Fresh BHI was used as a control. 500 mL of the samples were mixed with 250 mL TCA (trichloro-acetic acid) 60% to a final concentration of 20% (v/v) and incubated on ice for 1 h. After the incubation the samples were centrifuged at 12,470 g for 15 min and the supernatants were removed. The pellets were washed with cold denatured ethanol (200 mL of ethanol per pellet), centrifuged at 12,470 g for 10 min and then, ethanol was removed and the protein pellets were dried in a heat block for 20 min. After drying, 40 mL of 2X loading buffer. (1.25 M Tris-HCl pH ¼ 6.8, SDS (Sodium Dodecyl Sulphate), 2-mercapto-ethanol, glycerol, chromophenol blue) was added to each pellet, and pellets were cooked for 5 min in water. 20 mL of each sample was loaded on to a SDS-PAA gel (12.5%), which
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was in the SDS-PAGE device. The proteins were separated by applying 300 mA and 300 V in the device. The loading buffer was Tris–glycine 10% (v/v) (glycine, Tris, SDS, pH78.3) and was allowed to run for approximately 1 h. Finally, the gels were stained with Commasie Staining Solution. 2.5. Data analysis The experimental data (cell density data) were log-transformed and plotted as a function of time. The inactivation model of Geeraerd et al. (2000) was fitted to the data (Eq. (1)): expðkmax SiÞ NðtÞ ¼ Nð0Þexpðkmax tÞ ð1Þ 1 þ ðexpðkmax SiÞ1Þexpðkmax tÞ with N [CFU/mL] the cell population, N(0) [CFU/mL] the initial cell population, kmax [1/min] the maximum specific inactivation rate, Si [min] the shoulder period and t [min] the time. The time for the fourth decimal reduction, t4D, was calculated, based on the estimated model parameters (Eq. (2)) (Buchanan et al., 1993): t 4D ¼ Si þ 4
lnð10Þ kmax
In a next step, stationary E. coli cells were inactivated at 54 1C and 58 1C in these spent media. Figs. 1 and 3 summarize the inactivation curves for the different experimental set-ups. Population inactivation was followed until approximately 3 log (CFU/mL) (i.e., the detection limit). For all cases, duplicate samples revealed similar behavior. All inactivation curves consist of a log-linear part, in most cases preceded by a so-called shoulder phase. This shoulder represents a period during which the bacterial cells are able to resynthesize a vital component and death ensues only when the rate of destruction exceeds the rate of synthesis. In the linear part, the rate of destruction is much higher than the rate of synthesis, and the microbial population decreases exponentially (Geeraerd et al., 2000). Generally, microbial heat inactivation dynamics, i.e., heat resistance, was determined by the duration of the shoulder length, Sl, and/or the inactivation rate denoted by kmax and/or an increase of the time for the fourth decimal reduction, t4D (Table 1). It should be mentioned that the values of the shoulder length Sl did not show a systematic trend as this parameter is – as with the lag phase for growth – not only determined by the actual inactivation conditions but also determined by (small differences in) the preceding conditions (Table 1).
ð2Þ
3.1. Effect of the time temperature pre-history on the spent medium
Modeling of the survival data was performed with GInaFiT (Version 1.5), a freeware add-in for Microsofts Excel (Geeraerd et al., 2005). Graphical illustrations were generated in MatLabs Version 7.4 (The Mathworks, Inc., Natick, USA). The experimental data were analyzed statistically via a t-student test in order to verify the significant indifference (Po0.05) of replicate experiments and the significant difference of the model parameters for different experimental conditions (P40.05).
When the bacteria were inactivated in spent medium resulting from S1 and S2, an increased heat resistance was observed compared to unmodified (fresh) Brain Heart Infusion broth (uBHI) (see Fig. 1). More specifically, an extension of the shoulder, a lower inactivation rate and an increase of the time for the fourth decimal reduction can be observed at 54 1C and 58 1C (see Table 1, Fig. 1(a) and (b), respectively). The extent of the increased resistance is approximately the same for S1 and S2, i.e., inactivation curves for the two scenarios run in parallel. For S3 (tested only at 54 1C), a reduction of the inactivation rate and a slight increase of the time for the fourth decimal reduction, compared to that of uBHI, were observed (Table 1, Fig. 1(a)). Also, a difference can be observed between inactivation in spent medium of S1/S2 and S3. The deviation between these scenarios (P4 0.05) is (partly) due to the fact that no shoulder phase can be observed for Scenario 3. As a result, inactivation starts earlier compared to S1 and S2. After approximately 50 min, the inactivation curves for S2 and S3 seem to evolve at the same rate. Two aspects of the spent medium that might explain the increased heat tolerance were further investigated: (1) the
3. Results In the present research, the possible protective effect of spent medium, obtained after exposure to mild heat stress, on the heat resistance of stationary phase (non pre-stressed) E. coli K12 MG1655 cells, was tested. Spent medium resulted from three heat treatments: (a) heating from 30 1C to 55 1C at 0.14 1C/min (Scenario 1, S1), (b) heating from 30 1C to 42 1C at 0.14 1C/min (Scenario 2, S2) and (c) heating from 30 1C to 42 1C at 0.8 1C/min (Scenario 3, S3).
Fig. 1. Microbial inactivation curves at (a) 54 1C and (b) 58 1C in (n) unmodified BHI broth, (o) Scenario 1, (þ ) Scenario 2 and (X) Scenario 3.
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Table 1 Values of the inactivation rate, kmax, and standard errors (SE) for all conditions under study. Inactivation medium
Exp.
kmax [1/min] SE 54 1C
Unmodified BHI (pH 7)
1 2
Scenario 1
1 2
Scenario 2
1 2
Scenario 3
1 2
Scenario 1 pH 7.5 (adjusted with KOH)
1 2
Ultrafiltrated Scenario 1
1 2
BHI of pH 6 (adjusted with acetic acid)
1 2
Sl [min] SE
t4D [min] SE
58 1C
54 1C
58 1C
1.26 10 1 5.76 10 3 1.28 10 1 5.60 10 3
1.79 100 1.13 10 2 1.13 100 1.27 10 1
22.5 4.7 21.3 4.9
4.5 0.2 3.1 0.7
93.0 5.8 95.3 5.7
9.6 0.3 11.2 1.2
7.79 10 2 3.62 10 3 7.81 10 2 2.20 10 3
1.07 100 2.52 10 2 1.08 100 3.30 10 2
36.5 8.7 43.6 4.4
6.7 1.1 7.0 0.3
154.6 10.3 161.5 5.5
15.4 0.3 15.5 0.4
6.82 10 2 2.27 10 3 7.34 10 2 2.01 10 3
1.39 100 5.74 10 2 1.53 100 5.00 10 2
No shoulder
No shoulder
8.4 0.2 8.8 0.2
134.9 4.5 125.5 3.5
15.0 0.4 14.8 0.3
–
No shoulder
–
–
No shoulder
–
106.7 4.8 103.3 5.1
2
8.63 10 3.89 10 3 8.92 10 2 4.37 10 3 2
– 1
54 1C
58 1C
–
9.82 10 3.98 10 3 9.31 10 2 5.59 10 3
8.07 10 1.97 10 2 8.38 10 1 1.82 10 2
18.9 6.1 13.9 8.2
2.8 0.8 2.9 0.3
112.8 7.2 113.0 10.1
14.2 0.5 13.9 0.4
5.68 10 2 4.03 10 3 5.67 10 2 2.98 10 3
–
40.6 13.9 36.7 10.5
–
–
–
202.7 18.1 199.1 13.6
–
34.5 3.3 25.6 6.8
7.0 0.3 4.6 0.5
152.7 8.2 143.8 4.1
14.7 0.8 15.3 0.6
2
8.43 10 1.82 10 3 7.25 10 2 2.58 10 3
presence of (extracellular) proteins (Figs. 2 and 3), and (2) the reduced pH of the spent medium (approximately 6 compared to 7.5 for uBHI) (Fig. 3). 3.2. Effect of the (extracellular) proteins 3.2.1. Presence of (extracellular) proteins SDS-PAGE analysis of the three pre-treated media indicated the presence of a variety of proteins compared to the proteins present in uBHI (Fig. 2). In the case of S1 and S2, a protein band with a molecular weight of approximately 100 kDa was present (more pronounced for S2). Furthermore, protein bands with a molecular weight of 60 kDa, approximately 50 kDa (more pronounced for S2), and 37 kDa were observed. For S3, only a protein band of approximately 60 kDa was observed. However, this band is not as pronounced as it is for S1 and S2.
– 1
6.65 10 1.27 10 2 6.42 10 1 2.12 10 2
100 kDa 75 kDa 50 kDa 37 kDa
25 kDa 20 kDa C1
3.2.2. pH readjustment of spent medium In order to distinguish between the protein effect and the pH effect, stationary phase E. coli cells were added in spent medium of S1 with the pH readjusted to 7.5 (pH of uBHI) and inactivated at 54 1C and 58 1C. After pH adaptation, the stationary phase cells remain more heat resistant than cells inactivated in uBHI (Table 1, Fig. 3, P40.05). The increase in the bacterial tolerance is more pronounced at 58 1C. 3.2.3. Ultra-filtration of spent medium The protein effect on the bacterial heat resistance was studied in more detail by inactivating the stationary phase cells in ultrafiltrated S1 spent medium at 54 1C. The pH of the spent medium
S1
S2
S3
uBHI
S1(UF)
Fig. 2. Image analysis of the SDS-PAGE gel: (C1) the letter of the gel, (S1) Scenario 1, (S2) Scenario 2, (S3) Scenario 3, (uBHI) unmodified BHI broth and (S1þ (UF)) Scenario 1 after ultrafiltrarion.
remained equal to 6 after the ultra-filtration. The ultra-filtrated medium was analyzed via SDS-PAGE in order to check whether all the proteins were removed. As can be seen on the gel of Fig. 2, no protein bands were present in the ultra-filtrated spent medium of S1, i.e., the only remaining stressing factor was the low pH. Heat inactivation at 54 1C in spent BHI with and without the extracellular proteins was rather similar, i.e., removing the proteins did not significantly change the thermotolerance of E. coli (Table 1, Fig. 3(a)).
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Fig. 3. Microbial inactivation curves at (a) 54 1C and (b) 58 1C in (n) unmodified BHI broth, (o) Scenario 1, (þ) Scenario 1 at pH 7.5 (adapted with KOH), (X) Scenario 1 after ultrafiltration and (B) BHI of pH 6 (adapted with acetic acid).
3.3. Effect of acidity of the medium In a second step, the effect of low pH on the heat resistance of E. coli was studied. Heat inactivation experiments were performed in uBHI of pH 6 at 54 1C and 58 1C. The pH of the broth was adjusted with addition of 50% (v/v) acetic acid. Acetic acid was chosen for the acidification of the BHI as it is the main acid produced by E. coli during growth in a glucose rich environment (such as BHI). As can be seen in Table 1 and Fig. 3, increased heat resistance was observed when the bacteria were inactivated in acidified BHI, compared to the heat inactivation in unmodified BHI at pH 7.5 (P40.05). More specifically, at 54 1C heat resistance was increased, i.e., an extended shoulder, a reduced inactivation rate and an increase of the t4D, can be observed. Generally, the thermotolerance was approximately the same as observed when the bacterial cells were inactivated in spent medium of S1 (Table 1, Fig. 3 (a)). At 58 1C, the increased heat tolerance was even higher for the acidified uBHI, compared to the spent medium of S1, i.e., the inactivation rate is lower (Table 1). The duration of the shoulder and the t4D were approximately the same both for S1 and uBHI at pH 6 (Table 1, Fig. 3 (b)). These results seem to suggest that the pH effect dominates the extracellular protein effect.
4. Discussion The inactivation of stationary E. coli cells in spent medium, obtained after exposure to a mild heat stress, occured slower compared to fresh Brain Heart Infusion medium. Two aspect of the spent medium can be brought forward as an explanation for the increased heat resistance: (1) the presence of specific proteins in the spent medium, and (2) the slightly reduced pH of the spent medium. The spent medium was produced by growing E. coli under three different dynamic temperature conditions (S1, S2 and S3). For all three cases, the final temperature was situated in the superoptimal temperature region for E. coli, which triggered the heat shock response and, therefore, the production of specific (heat shock) proteins, which increase cell stability at higher temperatures. The inactivation in spent medium of S1 and S2
was almost identical, which suggests that the final temperature has no influence on the heat resistance. It is, however, most likely necessary that superoptimal temperatures (T437 1C) are reached such that the heat shock response is induced. Fig. 2 shows that the same proteins were present in the spent medium of S1 and S2 and that concentrations were in the same range, i.e., the intensity of the protein bands is similar. A small difference can be observed between inactivation curves of S1 and S2 – with heating rate 0.14 1C/min – and S3 – with heating rate 0.8 1C/min. More specifically, for S2 the inactivation rate was smaller and the shoulder phase and the time for the fourth decimal reduction longer, compared to S3 (Table 1, Fig. 3(a)). As the initial and final temperatures of these scenarios were identical (initial temperature: 30 1C, final temperature: 42 1C), this observation seems to suggest that the difference in the heating rate (S2: 0.14 1C/min, S3: 0.8 1C/min) results in different heat responses, which lead to different inactivation dynamics. A study of Tsuchido et al. (1982) showed that the slower E. coli K12 cells were heated from 0 1C to 50 1C, the higher the heat resistance of these cells was at 50 1C. Valdramidis et al. (2006, 2007) also indicated that, for a temperature up-shift from 30 1C to 55 1C, lower heating rates resulted in higher heat resistance of E. coli. Most likely, cells are more able to adjust to the environment and become more heat resistant when a slower temperature increase is imposed (Mackey and Derrick, 1987; Juneja and Marks, 2005). It is possible that in S2, i.e., with a slower heating rate (0.14 1C/min), the cells have more time to adapt to the rising temperature and, therefore produce more components or higher concentrations, compared to S3, which possibly results in an increased resistance of non pre-stressed cells. This hypothesis is confirmed by the SDS-page gel shown in Fig. 2: less or less pronounced protein bands are observed for S3, compared to S1 and S2. The molecular weights of some of the proteins found in the spent medium of S1 (Fig. 3) were similar to those of the most known HSPs (Ohtsuka et al., 2007; Rowburry, 2003; Juneja et al., 1998). Specifically the proteins of 100 kDa had similar molecular weight to the CIpA, ClpB, CIpC, CIpX and CIpY proteins of the HSP100 family, which contribute to the induction of thermotolerance (Ohtsuka et al., 2007). Generally, similar proteins can be recovered for S1 and S2, which resulted in almost identical
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inactivation kinetics, in contrast to S3 where less proteins were observed and inactivation occurred faster. This observation suggests that the presence of specific proteins somehow alerts or protects the non pre-stressed cells leading to an increased thermotolerance. These findings are in agreement with the conclusion of Heredia et al. (2009), who studied the involvement of extracellular compounds in the protection of Clostridium perfringens to heat, and showed that extracellular factors (proteins) produced by heat shocked cells have a thermoprotective function for non-stressed cells. Next to the presence of the proteins, the biggest difference – relevant for microbial inactivation kinetics – between fresh BHI and the spent medium was the pH. During the heat treatments, E. coli produced acetic acid, which resulted in a lower medium pH, i.e., 6, compared to approximately 7.5 of fresh BHI. This acidified environment was an additional stressing factor that may affect the (heat) resistance of E. coli. A series of inactivation experiments at 54 and 58 1C showed that the pH has a significant effect on the heat inactivation of E. coli. A first experiment, in which the pH of the spent medium (S1) was adapted to 7.5 (pH of uBHI), showed that the heat resistance was higher when compared to the uBHI, i.e., a slight prolongation of the shoulder (Table 1), a reduction of the inactivation rate and an increase of the t4D (Table 1) can be observed for both studied temperatures. The increase on the heat tolerance was more pronounced at 58 1C (Fig. 3). This finding is an indication that the presence of the proteins somehow increases the thermotolerance. In a second experiment, stationary phase cells were inactivated at 54 1C in ultra-filtrated spent medium (S1) at its original pH ( E6). By ultrafiltatrion, all the proteins present in the spent medium were efficiently removed (Fig. 2). The bacterial heat resistance did not alter after removing the proteins, i.e., inactivation dynamics were identical to the complete S1 spent medium. The observation that the thermotolerance remained at the same level in absence of the proteins is an indication that, most likely, the mainly low pH of the spent medium triggers the cells leading to an increased heat resistance. In a third series of experiments, the beneficial effect of the lower pH on the microbial heat resistance was confirmed. Stationary E. coli cells were inactivated in fresh BHI of pH 6 at 54 1C and 58 1C. The pH of the broth was readjusted with addition of 50% (v/v) acetic acid to mimic a real pH decrease as a result of microbial growth. Generally, increased resistance can be observed for the acidified fresh BHI compared to the uBHI (pH 7.5) and the S1 spent medium at pH 6 (Table 1, Fig. 3). Most likely, the different composition of the spent medium, i.e., less nutritional components, other (toxic) metabolic products and a different amount of acid, i.e., different pH level of the growth medium, resulted in a different microbial response to the stressful conditions. The increased heat resistance observed in the acidified fresh BHI corresponds with findings of other studies. Velliou et al. (2011) have shown that the heat resistance of E. coli at 54 1C and 58 1C increases when (pre-)exposed to different kinds of acids, i.e., acetic, lactic and hydrochloric acid. The extent of the resistance is dependent on the type of acid and on the quantity added. Acidification of the growth medium with acetic acid at a pH level of 6 and 5.5 shortly before heat inactivation led to an increased bacterial heat tolerance at 54 1C and 58 1C. Tetteth and Beuchat (2003) have proven that a heat and/or an acid shock can lead to an increase of the concentration of heat shock proteins, and finally to an increased acid tolerance of Shigella flexneri. According to Jorgensen et al. (1999), the presence of lactic acid has a specific positive effect on the response of Listeria monoytogenes to heat. Skandamis et al. (2008) observed that there is increased heat resistances of stationary phase cultures of L. monocytogenes at 52, 57 and 63 1C, following exposure to an acidic environment (acidified with lactic acid).
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5. General conclusions When stationary phase E. coli cells were heated in BHI under dynamic temperature conditions that reached the superoptimal temperature range, pH lowered due to acid production and heat shock proteins were produced as a response to the mild temperature stress. When unstressed stationary E. coli cells were inactivated in this spent medium, an increase in thermotolerance was observed. Based on a series of inactivation experiments, it can be concluded that this increased heat resistance resulted from both the presence of the heat shock proteins in the spent medium and the lowered pH. Experiments, which separate both effects, showed that mainly the lower pH resulted in the increased thermotolerance. Most likely, the presence of acetic acid induced an acid stress response, which resulted in a cross protection for the heat. This knowledge is of great importance in the food processing industry and could contribute in a more efficient design of emerging technologies and processes used in our days, in which milder heating conditions are applied compared to the conventional methods for achieving pasteurization.
Acknowledgments We would like to thank Prof. Jef Anne´ and Philip Gutschoven (Department of Microbiology and Immunology, Rega Institute, Katholieke Universiteit Leuven) for providing equipment and their help in the SDS-PAGE electrophoresis and Prof. Bart Van der Bruggen for kindly providing the ultrafiltration device in his laboratory. This work was supported by projects PFV/10/002 (Center of Excellence-OPTEC Optimization in Engineering) of the Research Council of the Katholieke Universiteit Leuven, project KP/09/005 (SCORES4CHEM) of the Industrial Research Fund and the Belgian Program on Interuniversity Poles of Attraction, initiated by the Belgian Federal Science Policy Office. E. Van Derlinden is supported by the postdoctoral grant PDMK/10/122 of the K.U.Leuven Research Fund. J. Van Impe holds the chair Safety Engineering sponsored by the Belgian chemistry and life sciences federation essenscia.
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