Abstracts. M. Cheroutre-Vialette 4

Effects of pH or a_w stress on growth of Listeria monocytogenes

M. Cheroutre-Vialette^*, I. Lebert, M. Hebraud, J. C. Labadie and A. Lebert

Meat Research Department, National Institute of Agronomic Research, 63122 Saint-Genès Champanelle, France

Received 25 November 1997; revised 15 January 1998; accepted 16 April 1998. Available online 2 July 1998.

ABSTRACT

The growth of three strains of Listeria monocytogenes at 20°C in a meat broth of different pH or water activity was investigated. At inoculation or at the beginning of the exponential phase, cells were exposed to stress by the addition of NaOH or NH₄⁺, acetic acid, NaCl or KCl, in order to reach a pH of either 9.0 or 5.6, or an a_w of 0.950 or 0.965, respectively. The effects of the exposure to stress on the generation and lag times of each strain were analysed by turbidity measurements for cultures in micro-titer plates. Results were confirmed by conducting the same experiments in a fermentor, except for the maximal population reached. The three strains showed similar behaviour. Cells were able to overcome the alkaline stress rapidly whereas acid and osmotic shocks induced important changes of the growth parameters. Cells exposed to acid or osmotic conditions from the time of inoculation were less affected than cells exposed at the beginning of the mid-exponential phase.

Author Keywords: Listeria monocytogenes; Growth parameters; Dynamic conditions; pH; Water activity

1. INTRODUCTION

Listeria monocytogenes, a psychrotrophic bacteria, is considered as an important food-borne pathogen. It can persist and grow at low pH (Farber et al., 1989 and Buchanan et al., 1993), high pH (Mendonca et al., 1994), low water activity (Nolan et al., 1992 and Marth, 1993) and at refrigeration temperatures (Walker et al., 1990).

Work on predictive microbiology has been carried out in order to improve the shelf life and safety of foods (Gould, 1989). Predictive models of microbial growths were set out with respect to the main controlling factors of the environment such as temperature, pH, and water activity. These factors were often considered constant during growth. But, environmental factors, particularly the temperature, may vary extensively throughout food processing. To take the environmental variations during time into account, different authors (Zwietering et al., 1994 and Van Impe et al., 1995) have improved predictive models and proposed dynamic models which describe growths at varying temperature profiles.

In this study, we report the effects of water activity and pH shifts on the growth of three strains of L. monocytogenes using different osmotic solutes (NaCl, KCl), organic acid (acetic acid) and bases (NaOH, NH₄⁺).

2. MATERIALS AND METHODS

2.1. Monitoring of bacterial growth

An automated turbidimeter (Bioscreen C, Labsystem, Labsystem France SA, Les Ulis, France) was used to follow the growth of Listeria strains in the micro-titer plates. Optical density (O.D.) was read at a wavelength of 600 nm.

A two-litre fermentor SET2M (Setric Genie Industriel, Inceltech, France) was used. The O.D. of the growth was measured with a spectrophotometer (UV-160A, Shimadzu, Japan) at 600 nm.

2.2. Microorganisms

The strains used by Bégot et al. (1997) in the studies with Bioscreen C were chosen for this work: L. monocytogenes CLIP 19804 isolated from meat products, L. monocytogenes 14 obtained from the food processing environment and L. monocytogenes CA Wisconsin associated with a cheese-borne listeriosis outbreak. All were serotype 4b which was involved in the recent epidemics in France (Rocourt et al., 1993). L. monocytogenes 14 strain was also used in the fermentor studies. Stock cultures were maintained on TSA agar (Difco, OSI, Maurepas, France) slopes and stored at 4°C.

2.3. Media

For preparation of inocula, cultures on TSA were subcultured in meat medium (MM) which contained meat peptone (Merck, Nogent sur Marne, France) 10 g/l, yeast extract (Difco) 5 g/l and glucose (Prolabo, Fontenay Sous Bois, France) 5 g/l.

The culture medium for growth studies was a tryptic meat broth (TMB) (Fournaud et al., 1973). It was buffered with a K₂HPO₄–KH₂PO₄ (Merck) 0.1 mol/l solution in proportion 1:1 (v/v) and adjusted to pH 7.0 with NaOH (40 g/l).

2.4. Types of experiment

For all experiments, three conditions were studied: standard, limiting and shock conditions. Under standard and limiting conditions, bacteria were grown in TMB or in TMB adjusted to the desired a_w or pH values (Table 1). The following additions were made to TMB: NaOH (solution of 200 g/l; Prolabo) 12.2 g/l or NH₄⁺ (Merck) 7 g/l to obtain pH of 9.0, acetic acid (Carlo Erba, Nanterre, France) 4.1 g/l to obtain a pH of 5.6, 80 g/l of NaCl (Prolabo) (Chirife and Resnik, 1984) or 80 g/l of KCl (Prolabo) (Jakobsen et al., 1972) to obtain a_w=0.95 and a pH of 7.0 or a_w=0.965 and pH of 7.0, respectively.

Table 1. Substrate additions and growth conditions
Full Size Table

The shock conditions were defined as follows: the bacteria were grown in standard TMB until the beginning of the exponential phase and were then exposed to the pH and a_w values described above. For this purpose, stock shock solutions of concentrations four times higher than indicated in Table 1 were prepared for use as described below.

2.5. Growth in microplates

Each strain was incubated on TSA slopes for 7 h at 37°C and then transferred to MM that was incubated in a rotary shaker waterbath (Aquatron, Infors, Switzerland) for 18 h at 20°C, by which time growth of all strains had reached the stationary phase.

A proportion of the culture was inoculated in the various media to give a concentration of about 5.10⁷ CFU/ml in order to be above the detection threshold of the Bioscreen C. For the inoculated TMB medium, 300 l were dispensed in 6 wells and the same volume of non-inoculated medium was dispensed in 4 wells in order to determine the O.D. of the growth medium and to detect possible contamination. The well numbers were considered as replicates. The same procedure was used for the pH and a_w adjusted TMB. The shock exposures were arranged as follows: 300 l of inoculated and 300 l of non-inoculated TMB were dispensed in 6 and 4 wells, respectively. An aliquot (100 l) of concentrated (×4) stock solution or standard TMB was added when the O.D. was near 0.2. The delay in adding the solutions to 70 wells was approximately 7 min. These plates were incubated in the Bioscreen C previously adjusted to 20°C.

2.6. Growth in fermentor

The protocol was similar to that in Bioscreen C except for the fermentor volume with additions adjusted accordingly. The additions lasted about 10 min. The experimental conditions were 20°C with a shaking speed of 100 rpm and an aeration regulated at 0.2 litre of sterile air per litre of medium per min.

2.7. Data analysis

Averages of the O.D. were calculated for the six repetitions of inoculated media and for the four repetitions of non-inoculated media. The data were then analysed using the procedure described by Bégot et al. (1996). Four quantities were calculated at time t:

A modified Gompertz equation (Zwietering et al., 1990) was used to fit the growth curves log₁₀ [(ΔO.D.)_t/(ΔO.D._min)]=f(t). Growth parameters such as A (logarithmic increase of population), L (lag time) and (maximal growth rate) were determined by non-linear regression with STAT-ITCF software (Gouet J.P. and Philippeau G., Institut technique des céréales et des fourrages, Paris, France). T_g (generation time) was derived from using the relation:

In standard and limiting conditions, the time of inoculation was taken as zero time when considering the growth curve. In shock conditions, the time of the addition of the shock solution was taken as zero time in the calculation of growth parameters.

3. RESULTS

The growth curves of L. monocytogenes 14 in the presence of acetic acid and NaCl can be seen in Fig. 1a and b, respectively. When the shock treatments (acid and osmotic) were applied after the beginning of growth of L. monocytogenes in standard media, lags were shorter, but generation times increased, as compared with cells grown in limiting media. This phenomenon was particularly pronounced for the acetic acid example (Fig. 1a).

Fig. 1. Comparison of growth curves observed in Bioscreen C with L. monocytogenes 14 when exposed to acetic acid (a) and NaCl (b) solutions under different conditions: limiting (- - -) and shock (———) conditions. Growth in standard TMB was represented by (– – –) according to the procedures used in the limiting condition and by (— – – — — – – —) according to the procedure used in the shock condition. The moment of solution administration for shock condition is represented by →.

The generation and lag times for each of the strains in all tested conditions are shown in Table 2. In comparison with growth in standard media, growth of the strain was not really affected by the presence of NaOH and NH₄⁺ whatever the treatment, limiting or shock conditions. The growth parameter values, i.e. generation and lag times, confirmed that cells adapted rapidly to the alkaline upshift to pH 9.0 with NaOH or NH₄⁺. Indeed, these values calculated in alkaline media were consistent with those found in standard media.

Table 2. The generation time (T_g) and lag time (L) in hours of Listeria strains grown under three conditions in micro-titer plates (Bioscreen C) at 20°C

Under acid and osmotic conditions, cell growth was more affected. Media containing acetic acid was the most severe culture condition for the growth of the three strains. Under limiting conditions, although the presence of acetic acid did not cause increasing lag times, generation times were at least 3.5 times greater compared to the values in standard media. The solute used to control a_w can influence the growth pattern of the microorganism. At the tested concentration (80 g/l), L. monocytogenes which is also considered as a salt-tolerant microorganism, tolerated KCl better than NaCl. Indeed, the generation and lag times in limiting and shock conditions, were higher in the presence of NaCl 80 g/l. This influence of NaCl can be due to a larger a_w effect.

Generally L. monocytogenes 14 and 19804 had similar generation times whereas L. monocytogenes CA had longer generation times under limiting conditions. Variations in lag time were noted among the strains in all tested conditions.

Fig. 2 shows the growth curves of L. monocytogenes 14 obtained for tested media in the fermentor. Similar effects of shock treatments were observed in the fermentor and in the Bioscreen C. The alkaline treatment had negligible effect on L. monocytogenes 14 (results not shown), whereas growth and lag phases were affected by acid (Fig. 2a) and osmotic (Fig. 2b) treatments. The strain was more affected by the presence of acid than by the presence of NaCl, i.e., the generation times were longer. The behaviour difference between limiting and shock conditions was also observed. For all treatments, the same population increase was observed under limiting and shock conditions. A difference was noted with the Bioscreen C for acetic acid treatment (Fig. 1a). The shock effect was amplified when cells were stressed in exponential phase. In this condition, the increase in the population was lower than in the limiting condition and lower than in the culture carried out in the fermentor.

Fig. 2. Comparison of growth curves observed in the fermentor with L. monocytogenes 14 when exposed to acetic acid (a) and NaCl (b) solutions under different conditions: limiting (- - -) and shock (———) conditions. Growth in standard TMB was represented by (– – –) according to the procedures used in the limiting condition and by (— – – — — – – —) according to the procedure used in the shock condition. The moment of solution administration for shock condition is represented by →.

Generation and lag times of cultures in the fermentor are presented in Table 3. The values of generation times were similar to those obtained in Bioscreen C, but large variations in lag phases were noted. The results obtained from the fermentor culture experiments confirmed the observations made with the Bioscreen C.

Table 3. The generation time (T_g) and lag time (L) in hours of L. monocytogenes 14 grown under different conditions in the fermentor at 20°C

4. DISCUSSION

In previous studies about growths in non-variable conditions, the interest in using the Bioscreen C for analysis of microbial growth parameters (Korkeala et al., 1992) and its good reproductivity (Bégot et al., 1996) was established. Our study confirmed these observations. The results obtained from the experiments carried out in the automated turbidimetric system were in agreement with those obtained in the fermentor. The increase in the number of L. monocytogenes was underestimated in the Bioscreen C as compared to the fermentor. The homogeneous aeration and agitation which were controlled in the fermentor might be a possible explanation for this observation. Nevertheless, the differences found in the generation times between the two methods were not significant. This suggests, therefore, that the Bioscreen C is suitable for studying the growth of bacteria under variable conditions. A high concentration of cells is required in the Bioscreen C for a detectable change in absorbance, but Buchanan and Phillips (1990) and Dalgaard et al. (1994) demonstrated that the growth kinetics of L. monocytogenes were unaffected by the size of the initial inoculum i.e., the generation and lag times derived from the Gompertz equation were not affected.

The L. monocytogenes strains showed similar responses following a_w or pH stresses. Variations were generally larger in lag times than in generation times. There was very little difference in generation times between L. monocytogenes strains 14 and 19804 for most of the experiments. Any major variations were observed with the most severe culture conditions, i.e., in the presence of acetic acid. L. monocytogenes strain CA was more affected by the different stress conditions and in particularly under limiting conditions. These findings are consistent with those of Papageorgiou and Marth (1989) who also reported that L. monocytogenes CA was less salt tolerant than strains in whey and skimmed milk containing 12% NaCl.

In recent years, the interest in developing dynamic mathematical models that describe the growth of microorganisms in the presence of temperature variations, has increased. When building dynamic models to predict bacterial growth with change in temperature (Zwietering et al., 1994), two hypotheses have been formulated: (i) the bacteria show no additional lag phase due to a change in temperature during the exponential phase, and (ii) growth continues immediately at the specific growth rate associated with the temperature post-shift. More recently, for the elaboration of dynamic models, Van Impe et al. (1995) and Rosso (1995) accepted as a postulate, that variable environmental conditions do not induce stress conditions on a microbial population, i.e., they considered that the microflora responds instantaneously to pH (Rosso, 1995) or temperature changes (Van Impe et al., 1995 and Rosso, 1995). Our tests carried out with the three L. monocytogenes strains showed that growth under modified osmotic or acid environmental conditions in exponential phase did not agree with these hypotheses. Since 1992, Van Impe et al. (1992) have recognized the need to take into account the previous history of a product, and therefore the previous history of a strain which could have an important impact on the lag time prediction of a microorganism. It is also necessary to quantify the ability of a microorganism to grow under variable conditions. This could allow the optimal combination of temperature, pH, a_w and other factors with the purpose of increasing the shelf life of a given product.

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Effects of pH or aw stress on growth of Listeria monocytogenes