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International Journal of Food Microbiology, 1998 Jan, vol. 39 (1-2), pp. 53-60

Growth of Pseudomonas fluorescens  and Pseudomonas fragi  in a meat medium  as affected by pH  (5.8 – 7.0),  water activity  (0.97 – 1.00)  and temperature (7 – 25°C)

I. Lebert, C. Begot and A. Lebert

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

Received 20 January 1997; revised 30 July 1997; accepted 21 October 1997. Available online 25 February 1998.



ABSTRACT 

A total of 59 strains of Pseudomonas, isolated from meat products, were grown in micro-titer plates in a meat medium over a range of pH (5.8–7.0), a_w (0.97–1.00) and temperature (7–25°C). Growths were performed in a meat broth with an automated turbidimeter (Bioscreen C, Labsystem, France). The growth curves obtained in this study did not have sigmoidal shapes making it impossible to calculate growth parameters using the Gompertz equation. The medium was weakly oxygenated in the micro-titer plates and reached 0%-dissolved oxygen at the beginning of the exponential phase. Strains were separated into two groups: P. fragi and P. fluorescens. Strains of P. fragi had shorter lag times than those of P. fluorescens. The impact of such results is interesting in that these could assist to explain the succession of flora that is observed during the processing of meat: P. fluorescens is the dominant bacteria among Pseudomonas spp. at the beginning of a slaughter line and P. fragi becomes dominant during the chilling process.

Author Keywords: Pseudomonas fragi; Pseudomonas fluorescens; Growth; Meat broth; Bioscreen C; Aeration
 

1. INTRODUCTION

Many flora of spoilage-bacteria have an effect on the shelf-life of refrigerated food products. The main flora responsible for spoilage in fresh meat and milk products during aerobic storage however are the Pseudomonas species. These are dominant in poultry meat (Pooni and Mead, 1984), pork (Coates et al., 1995), and beef and lamb (Widders et al., 1995). In milk and cream the major spoilage bacteria have been identified as P. fluorescens and P. fragi (Champagne et al., 1994). Widders et al. (1995) reported that the average contamination on whole carcasses at 4°C peaked at 3.90 log₁₀ cfu/cm² and 4.54 log₁₀ cfu/cm² on meat surfaces at the same temperature. P. fragi and P. fluorescens cause deterioration in quality of meat and milk products due to the production of extracellular proteases and lipases at low temperatures. Off-odours occur in milk when the population of Pseudomonas spp. reaches 2.2×10⁶ to 3.6×10⁷ cfu/ml (Champagne et al., 1994) or on meat when the population reaches 10⁷ to 10⁸ cfu/cm². P. fragi strains produce fruity and putrid odours on beef (Dainty et al., 1989) and have a deleterious effect on the colour of meat stored at 1°C, resulting in a green and slimy appearance (Bala et al., 1977).

Although many studies have been carried out on the occurrence and levels of contaminations in meat products (Molin and Ternström, 1982Gustavsson and Borch, 1993Coates et al., 1995Widders et al., 1995), there has been little work on a comparison of relative growth of Pseudomonas species and on the growth variations among these strains. The aim of this study therefore was to compare the growth rates of strains of P. fragi and P. fluorescens in a selected media over a range of pH, water activity (a_w) and temperature, and to attempt to describe the resulting growth curves.

2. MATERIALS AND METHODS

2.1. Strains

A total of 59 strains of Pseudomonas were selected from bacterial counts made from meats of different origin(Table 1). All were isolated from or grown on Pseudomonas Agar Base (Oxoïd, Unipath Ltd., Basingstoke, UK) supplemented with Pseudomonas CFC Supplement (Oxoïd). The strains were identified as P. fluorescens or P. fragi using standard tests (Molin and Ternström, 1982). This involved: growing on Plate Count Agar (PCA; Interbio, Comptoir Lyonnais de Verreries, Villeurbanne, France) at 4°C, and a range of tests including Gram stain, oxidase and catalase reaction, mobility, production of acid in the presence of maltose and cellobiose, degradation of gelatine and production of pigment on King B medium (Merck, Nogent sur Marne, France). The strains were stored at 4°C on PCA slants and transferred monthly.

 

 

 

Table 1. List of Pseudomonas strains studied and their source
 

 

2.2. Media

Two media were used for successive subcultures — these were PCA slants and meat medium (MM) which contained meat peptone (Merck) 10 g/l, yeast extract (Difco, OSI, Maurepas, France) 5 g/l, and glucose (Prolabo, Fontenay sous Bois, France) 5 g/l. The pH was adjusted to 7.0 by NaOH (40 g/l). The culture medium was a tryptic meat broth (TMB) (Fournaud et al., 1973) and was buffered with a K₂HPO₄-KH₂PO₄ (Merck), 0.1 mol/l solution to adjust the pH. Water activity was controlled by adding NaCl (Merck) according to Chirife and Resnik (1984). An aliquot of 0.5 ml/l of anti-foam ANBIO 15 (1% v/v) (Erol, Ouvrie, Lesquin, France) was added for growth in a fermenter.

2.3. Materials

An automated turbidimeter (Bioscreen C, Labsystem, Labsystem France SA, Les Ulis, France) was used to follow the growth of Pseudomonas spp. in the micro-titer plates. Optical density (OD) of the growth was measured on a spectrophotometer (UV-160A — Shimadzu Corporation, Japan). OD was read at a wavelength of 600 nm. The threshold of detection for the Bioscreen C, and for the spectrophotometer, were determined at 6.0×10⁶ cfu/ml and 1.0×10⁷ cfu/ml, respectively (Bégot et al., 1996). The range where OD was proportional to the bacterial population extended from 0.010 to 0.8 OD for both apparatus.

A 2-l fermenter (SET2M, Setric Génie Industriel, Inceltech, Toulouse, France) was used with pH monitored by a heat-sterilisable electrode (Ingold, Inceltech, Toulouse, France). Dissolved oxygen was measured directly in the fermenter with an oxygen probe (Ingold) and the zero calibration determined with an oxygen-free medium provided by Setric Génie Industriel. A full-scale reading was obtained by saturation of the medium with air supplied continuously to the culture.

2.4. Inoculum

Each strain was incubated on PCA slants at 25°C for 8 h and then transferred to MM that was shaken in a rotary shaker waterbath (Aquatron, Infors, Massy, France) for 17 h at 25°C, by which time growth of all the strains had reached the stationary phase. A proportion of the culture was inoculated in TMB to give a concentration of about 10⁷ cfu/ml in order to be above the detection threshold of the spectrophotometer and the Bioscreen C. The viable number of bacteria was determined on PCA plates using standard microbiological practice immediately after inoculation of the growth medium. Viable numbers for growth in Bioscreen C ranged from 6.9–7.1 log₁₀ cfu/ml and from 7.1–7.3 log₁₀ cfu/ml for growth in fermenter.

2.5. Growth experiments

Growth of the strains was obtained on micro-titer plates using the protocol developed by Bégot et al. (1996): for each strain, 300 l of inoculated TMB was dispensed in eight successive cuvettes in the micro-titer plates. Some cuvettes were filled with non-inoculated TMB and were used as controls. OD was measured at 600 nm.

The working volume of the fermenter was 1 l. The stirring speed was regulated to 70 rev./min. Prior to inoculation the medium was saturated with oxygen, so that growth began at a level between 90 and 95% of dissolved oxygen. Air could be supplied at the following volumetric rates: 0, 0.24, 0.40 and 0.80 l/l of medium/min. A fraction of the fermenter volume was periodically removed to measure OD, however, no more than 10% of the working volume was pipetted during growth.

2.6. Growth conditions

A fractional factorial design for each of the environmental variables, a_w, temperature, pH at three levels, gave nine conditions for growth for each of the 59 Pseudomonas strains. These growth conditions are given in Table 2. Growth in the fermenter was carried out with P. fragi 103 in condition E111.

 

 

 

Table 2. Conditions of a_w, temperature and pH in the fractional factorial design

 

2.7. Growth curve treatments

Growth data obtained as a function of OD versus time were transferred from Bioscreen C to Excel software (Microsoft Windows) and transformed for each measurement. Four quantities were calculated at time t (Bégot et al., 1996):

 

1. (ODi)t, the OD average for the eight replicates

 

2. (ODni)t, the OD average for the non-inoculated TMB

 

3. (ΔOD)t=(ODi)t−(ODni)t

 

4. f(t)=Log₁₀[(ΔOD)t/ΔODmin] where ΔODmin was the lowest ΔOD value above the detection threshold.

The function f(t) was used to calculate two parameters for the 59 strains in the nine conditions: the maximal growth rate, V_max, and the lag time, L. The maximum growth rate of all the growth rates V_max, was calculated for each time t_n (Eq. 2). L corresponded to the intersection of the x-axis and the tangent in V_max:

GT was the generation time when V was maximal:

2.8. Statistical analysis

Average, mean, minimum and maximum were calculated for the two variables L and GT. Strains were classified according to their ability to grow in the different conditions. A cluster analysis (Everitt, 1980) was carried out with STAT-ITCF software (J.P. Gouet and G. Philippeau, Institut Technique des Céréales et des Fourrages, Paris, France).

3. RESULTS

Strains of both species grew at 4°C on PCA, were Gram-negative, motile, gave positive oxidase and catalase reactions. P. fluorescens strains produced fluorescent pigments and gelatinase, P. fragi strains did not. P. fragi strains produced acid in the presence of maltose and cellobiose, P. fluorescens strains did not.

As shown in numerous studies, a decrease in temperature, pH or a_w increased the duration of both lag and generation times. Fig. 1 shows for most growth conditions, growth was characterised by an unusually shaped growth curve, i.e. the lag phase was clearly visible with the growth phase generally reaching its maximal growth rate at the beginning of this phase. After the maximal growth rate, there was a break in the curve leading to a decreasing phase which did not reach a steady stationary phase, even after long periods of incubation (up to 45 h in condition E111). These growth curves are thus characterised by an inflection point situated not long after the end of the lag phase and by a non-symmetrical shape. The two parameters GT (generation time) (Eq. 3) and L (lag time) are shown in Table 3 and Table 4, respectively. It is seen that the interval between minima and maxima increased as the conditions of pH, a_w and temperature moved away from the most favourable condition E111. It is to be noted that the gap between minima and maxima increased more for L than for GT.

 

 

Fig. 1. Comparison of growth of () P. fragi 11×11 and () P. fluorescens 19.6B in conditions E213 (a_w 0.985, 25°C, pH 5.8).

 

 

Table 3. Comparison of averages, means, minima and maxima obtained for the generation time (h) in all the conditions tested, on all the strains
 

 

 

Table 4. Comparison of averages, means, minima and maxima obtained for the lag time L (h) in all the conditions tested, for all the strains and for both groups obtained after the classification. (The first group was made up of 40 P. fragi and four P. fluorescens, the second of 15 P. fluorescens)
 

 

Fig. 2 shows that P. fragi 103 rapidly metabolised dissolved oxygen when no air was supplied, i.e. there is a break in growth as the dissolved oxygen level reached 10% 1.5 h after inoculation. The time needed to reach 10% of the saturated value for dissolved oxygen was 1.5, 1.5, 2.5 and 3.5 h when the rates of air were respectively 0, 0.24, 0.4 and 0.8 l per l of medium per min. Zero percent dissolved oxygen was reached 1 h following these periods. Growth curves in these four conditions are shown in Fig. 3. For 2 h, growth curves are similar, then they separated and rose faster as the air supply increased. The growth curve obtained in Bioscreen C was close to that obtained in the fermenter at a flow rate of 0.24 l per l of medium per min. It assumed confidently that conditions of aeration in the Bioscreen C cuvettes are similar to this type of aeration.

 

 
 

Fig. 2. Growth of P. fragi 103 (–  –) in fermenter in condition E111 (a_w 1.0, 25°C, pH 7.0) and evolution of dissolved oxygen (———). No air was supplied to the medium as it was inoculated.

 

Fig. 3. Comparison of growth of P. fragi 103 in condition E111 (a_w 1.0, 25°C, pH 7.0). Growth was studied in Bioscreen C (– × –) and in a fermenter at air flow rates of 0 (–  –), 0.24 (–  –), 0.4 (– ♦ –) and 0.8 (–  –) l of air per l of medium and per min.

 

The classification carried out on all the strains revealed two main groups (Fig. 4). The first was comprised of all the P. fragi and four P. fluorescens namely 51, 11.6A, M2L3 and P×10. The second comprised the remaining P. fluorescens. Statistical values for all strains are presented in Table 3. Table 4 presents L, averages, means, minima and maxima for each group. It can be seen that shorter lag times were obtained for the first group. Between each group differences were greater in conditions E133 (a_w 1.00, 7°C, pH 5.8), E232 (a_w 0.985, 7°C, pH 6.4), E331 (a_w 0.97, 7°C, pH 7.0) related to the lowest temperature.

 

 

Fig. 4. Dendrogram of the cluster analysis carried out on the lag times calculated for the 59 Pseudomonas strains in the nine conditions of the experimental design.

 

4. DISCUSSION

The growth curves obtained in this study did not have sigmoidal shapes making it impossible to calculate growth parameters with the Gompertz equation which is usually used by authors (Zwietering et al., 1990; Buchanan and Klawitter, 1992; Dalgaard et al., 1994 and McClure et al., 1994). A steady stationary phase was rarely obtained and for long experiments a biofilm could be seen on the top of the medium in the cuvettes. In addition to possible insufficient shaking of the micro-titer plates in Bioscreen C (Laplace et al., 1993), it was supposed that the aeration was also too weak. Growth in the fermenter confirmed this and highlighted that the aeration in the cuvettes was equivalent to 0.24 l of air per l of medium per min. These conditions of aeration played too important a role for aerobic bacteria and became a limiting factor for the growth of the Pseudomonas spp. A possible explanation for the unexpected shape of the growth curves is that the combined effect of the depletion of oxygen and the change in the metabolism: indeed, in some cases, Pseudomonas spp. can use nitrate as an alternate electron acceptor and can grow anaerobically (Palleroni, 1984).

For many studies carried out in milk and milk products with Pseudomonas spp. the generation time was the main parameter for comparison (Shelley et al., 1986 and Marshal and Schmidt, 1988). Cox and MacRae (1988) reported that P. fragi grew faster than P. fluorescens in both U.H.T. and raw milk at 4°C. In meat products, few studies have compared the behaviour of the species. This work carried out on 59 strains of Pseudomonas has shown small differences between calculated generation times that are confirmed by results of Pooni and Mead (1984) who worked on strains isolated from poultry products and grown in heart infusion broth. These authors showed that pigmented and non-pigmented Pseudomonas made little difference in average generation times. The differences in lag time observed between both species are important. For the 59 strains separated into the two groups, only four strains of P. fluorescens were in the P. fragi group. These were characterised by shorter lag times in condition E111 (25°C, a_w 1, pH 7). This explains their presence in the first group. The P. fragi group had on average shorter lag times than P. fluorescens. These differences have been observed on a great number of strains and in a wide range of temperature, pH and a_w; e.g. average L was two-fold shorter in the P. fragi group at 7°C.

These results could help to explain the changes in the ecology of Pseudomonas flora shown by different authors who have studied levels of contamination and areas of isolation during meat processing and retailing. Lahellec and Colin (1981) observed the spoilage strains in poultry: pigmented strains represented the major population isolated in processing plants, but during storage non-pigmented strains outgrew the former. On beef, lamb and pork, studies have shown the predominance of P. fluorescens from the slaughter line to the chilling process (see e.g. Gustavsson and Borch, 1993). P. fluorescens is known to be largely present in the environment (floor, water), on animals (hide, skin) or also in water and surfaces in meat factories (Drosinos and Board, 1995). On cutting lines and during storage and retailing, P. fragi was then found as the dominant flora on meat (Molin and Ternström, 1982Molin and Ternström, 1986Prieto et al., 1992). Drosinos and Board (1995) studied the evolution of the bacterial flora in minced lamb stored aerobically at 4°C and showed that P. fragi outgrew P. fluorescens. They tried to explain this succession of flora by differences in the metabolism of both species. In our study shorter lag times observed for P. fragi strains could be linked to their ability to adapt better to new environmental conditions compared to P. fluorescens.

Our findings make it possible to choose a limited number of representative strains from the 59 Pseudomonas — so as to diminish the number of experiments needed to study these few strains with more precision in broth or on surface tissue of meat. A ‘low', ‘quick' or ‘average' strain characterised by a slow, rapid or average growth in all or few conditions of the experimental design, could then be chosen in both species. Further studies should be undertaken: to substantiate the differences in lag times between the ‘average' strains of both species and compare their generation times in better conditions of aeration; to study a ‘low' strain and a ‘quick' strain and observe the variability in their growth responses; or to compare the growth parameters obtained in broth and on the surface tissue of meat.

ACKNOWLEDGEMENTS

This work was supported by four food companies, collaborating in a research Program UNIR (Ultra-propre Nutrition Industrie Recherche), the French Research Department and the French Agricultural Department. We are sincerely grateful to the laboratories which provided bacterial strains. We wish to thank Dr. K.R. Davey for his help in the revision of the manuscript.
 

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