The effect of irradiation and pH on Lactobacillus curvatus

International Biodeterioration & Biodegradation 32 (1993) 75-85

The Effect of Irradiation and p H on Lactobacillus curvatus

L.M. Fielding, P.E. Cook & A.S. Grandison Department of Food Science and Technology,Universityof Reading, Whiteknights, Reading, UK, RG6 2AP

ABSTRACT

Lactobacillus spp. display a higher radiation resistance than most bacteria and can withstand doses o f between 2.5 and 5.0 kGy. This leads to their dominance in the microflora o f radurised meats. The lactobacilli are also acid-tolerant, with a p H range for growth of 7.2-3.0. As the severity of radiation processing may be reduced by combination with other treatments, the effect o f irradiation on Lactobacillus curvatus was examined over a range of p H values. Exponential phase cultures of L. curvatus, containing approximately 10s cells m1-1, were irradiated in de Man, Rogosa & Sharpe broth at doses o f 0-1.9 kGy, at p H values ranging from 7.5 to 4.0. Lowering the p H from 4.6 to 4.3 and below before irradiation had no effect on the survival rate of L. curvatus up to a dose of 1.9 kGy. An increase in p H to 7.0 before irradiation led to a marked decrease in survivors, over a dose range of 1.0-1.9 kGy. No further decrease was seen when the p H was increased to 7.5 prior to irradiation. This effect was only observed when the p H was altered before radiation processing.

INTRODUCTION The main purpose of food irradiation is to prolong the shelf-life of a product by reducing the number of spoilage organisms and destroying potential pathogens (Urbain, 1986, p. 118; Patterson, 1988). The process of radicidation is an ionising radiation treatment intended to eliminate all non-sporeforming organisms of public health significance. The doses used are in the region of 3 kGy (Josephson & Peterson, 1982). 75 International Biodeterioration & Biodegradation 0964-8305/94/$07.00 q 1994 Elsevier Science Limited, England. Printed in Great Britain.

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L.M. Fielding, P.E. Cook, A.S. Grandison

Lactobacillus spp. are relatively resistant to irradiation and it has been reported that they can withstand doses of up to 5 kGy (Hastings et al.,

1986; Patterson, 1988). Radicidation will, therefore, result in a number of these organisms surviving and becoming dominant over the less resistant pathogens. The food will become spoiled before it presents a health hazard, as lactobacilli are rarely pathogenic (Brock et al., 1970). The severity of radiation processing can be reduced by combination with other treatments, thus ensuring the microbiological quality of the food without compromising its sensory properties (Urbain, 1986, p. 257; Anon., 1991). Processes such as irradiation can injure cells at doses well below the inactivation dose. It is, therefore, logical to apply other stresses to injured cells to further damage or destroy them (Gould, 1989). The lactobacilli are also more acid tolerant than most pathogens, having a pH range for growth of 7-2 to 3.0 (Jay, 1978). The aim of this work was to determine whether Lactobacillus curvatus maintains its resistance to irradiation and conditions of low pH when these treatments are applied in combination. This is important, as Lactobacillus spp. have the potential for providing a 'fail-safe' during storage of irradiated foods.

MATERIALS A N D M E T H O D S Organism and media

Exponential phase cultures of L. curvatus NCFB 1041 were used. In all experiments the organism was suspended in de Man, Rogosa & Sharpe broth (MRS; Oxoid CM359), prepared with high-purity water (Purite R O 5 0 reverse osmosis unit and HP deionisation column). The exponential phase culture was prepared by diluting an 18-h subculture 100-fold, in buffered peptone water (BPW; Difco 1810-17-9). One millilitre was placed in a 250-ml conical flask containing 90 ml of MRS broth (pH 6-23), and was shaken at 250 rpm at 30°C for 9 h (pH 6.12). The culture was refrigerated overnight (pH 4.81), and exponential growth was resumed by shaking, as before, for 1 h before use (pH 4.63). Plate count method

Enumeration of survivors was determined, in all experiments, by means of pour plates. Tenfold dilutions of the test culture were prepared in BPW, and 1.0 ml of the desired dilution was added, in duplicate, to sterile petri dishes. The medium for enumerating bacteria was prepared by the addition

Irradiation and p H effect on Lactobacillus curvatus

77

of 1.2% agar (Oxoid, LI3) to M R S broth prior to autoclaving. All plates were incubated at 30°C for 48 h. Only plates containing between 30 and 300 colonies were counted. Labsystems Bioscreen

The Bioscreen (Labsystems, Basingstoke) is an automated microbial growth analyser which, by means of vertical light photometry, measures the absorbance of multiple samples over a period of time. As the absorbance of a bacterial suspension is directly proportional to the number of organisms present, a growth, or turbidometric curve may be obtained. The absorbance of the samples was measured every 10 min, at a wavelength of 600 nm, for a period of 48 h. After each absorbance reading, the plate was shaken automatically. A control sample, consisting of uninoculated MRS broth, was also measured to determine the stability of the absorbance signal. The parameter used to analyse the Bioscreen results was the time taken for a culture to reach a given absorbance value. As the number of cells present in the sample decreases, the time taken for the culture to reach this absorbance increases. It is possible to quantify injury using this method, as stressed cells may operate repair mechanisms, thus displaying an extended lag phase. If the initial cell number is known, the lag phase extension can be calculated and used as an index of injury. The radiation source

Electron beam irradiation, produced by a Van de Graaff accelerator, was used. The accelerator had an energy range of 0.5-2-0 MeV with a beam current of 0-50#A. The sample was placed on a conveyor belt which passed beneath a scanning horn, resulting in an even dose distribution. The conveyor speed and duration of irradiation were, as far as possible, kept constant. The dose was raised by increasing the beam current, and the dose absorbed was measured using optichromic dose meters (Far-West Technology FWT-70-83, Goleta, CL, USA). The effect of pH

Five pH values were chosen (7.0, 4.6, 4.3, 4-13 and 4-0), corresponding to equal increments of hydrogen ion concentration. For each pH value, 5 ml of double-strength M R S broth (pH 6.1) was modified to the desired pH using 0.1 M hydrochloric acid or 0.1 M sodium hy,droxide, and the volume

78

L.M. Fielding, P.E. Cook, A.S. Grandison

was made up to 10 ml using sterile, high-purity water. Ten millilitres of the exponential culture were centrifuged at 5000 rpm for 5 min (IEC Centra4X Bench Top Centrifuge) and the cells were resuspended in the modified broth. After incubation at 25°C for 1-5 h, I-0 ml was transferred to a further 9 ml of pH-modified broth. A Bioscreen plate was prepared (six replicates per sample, 0.4 ml of inoculum per well), and plate counts were carried out.

The effect of irradiation The exponential culture was centrifuged, as before, and the cells were resuspended in standard M R S broth. F o u r millilitres of the culture were placed in each of two wells of a six-well, fiat-bottomed plate (Sterilin 33F06L). The culture was irradiated at doses of 0-3.2 kGy. Immediately after irradiation, a tenfold dilution was made in MRS broth. A Bioscreen plate was prepared and plate counts were carried out as before.

The effect of modified pH followed by irradiation The exponential culture was centrifuged and the cells were resuspended in pH-modified MRS broth. After incubation at 25°C for 1.5 h the culture was irradiated, in a six-well plate, at doses of 0-1.9 kGy. Immediately after irradiation, a tenfold dilution was made in pH-modified broth. A Bioscreen plate was set up and plate counts were carried out as before.

The effect of irradiation followed by modified pH The exponential culture was centrifuged and the cells were resuspended in standard M R S broth. After irradiation, in six-well plates, at doses of 01.9 kGy, a tenfold dilution was prepared in pH-modified broth. The cultures were incubated at 25°C for 1.5 h. A Bioscreen plate was set up, pour plates were prepared, and counts were carried out as before.

RESULTS

Modified pH The exposure of L. curvatus to conditions of modified pH, over a range of pH values between 7.0 and 4.0, had little effect on the survival of the organism over a period of 1.5 h. Prolonged exposure at pH 4.6 and below, however, led to a decrease in the growth rate of the organism, and to a

Irradiation and p H effect on Lactobacillus curvatus

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decrease in the absorbance reached after approximately 2000 min (33 h) incubation. On increasing the p H to 7.0, no decrease in growth rate or final absorbance was seen (Fig. 1). Irradiation Figure 2 shows that the number of organisms surviving the radiation treatment was proportional to the dose absorbed by the liquid culture. There is no apparent shoulder or tail effect (areas of increased resistance at the extremes of the survival curve) over the dose range used. No growth was seen after a dose of 3.2 kGy. Linear regression analysis was performed to determine the dose required for a 90% reduction in survivors (the Dl0 value) using the reciprocal of the slope. This was found to be 0-36 kGy. As the dose absorbed increased, the apparent duration of the lag phase of the culture also increased (Fig. 3). This p h e n o m e n o n could be due to a decrease in the number of viable organisms present, to repair of injured cells, or to a combination of these factors. The number of survivors was compared with the time taken for those survivors to reach a

80

L.M. Fielding, P.E. Cook, A.S. Grandison

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Irradiation and p H effect on Lactobacillus c u r v a t u s

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predetermined absorbance value, and these data were compared with a calibration curve prepared using, dilutions of non-irradiated culture (Fig. 4). It has been shown, using analysis of variance, that the comparative slopes of the two lines are significantly different (calibration line gradient, -0-401; irradiation line gradient, -0-283).

Modified pH followed by irradiation Lowering the pH from 4.6 to 4-3 and below, before irradiation, had no additional effect on the number of survivors, but an increase in pH to 7.0 and 7.5 led to a greater decrease in cell number (Fig. 5). This effect was seen at doses of 1-05 kGy and above. The recovery of L. curvatus at pH values of 7.0--4.0 after 0.35 kGy irradiation resembles the recovery of the organism after no irradiation, i.e. a decrease in growth rate and final absorbance as the pH value decreased. At a dose of 1-05 kGy however, the growth at pH 7-0 was negligible (Fig. 6).

Irradiation followed by modified pH There was little effect on the survival of the organism when the pH was modified after radiation processing (Fig. 7). The recovery pattern of all irradiated cells was similar to the trends shown in Fig. 6 (modified pH followed by irradiation). The most notable of these is the minimal growth at pH 7-0.

DISCUSSION Patterson (1988) reported that the D~0 value for Lactobacillus sp. was 0.593 kGy when irradiated in chicken mince. The value found here, 0-35 kGy, was obtained by irradiation in M R S broth. The difference in the values is explained by the fact that food components exert a protective influence on the bacteria (Dyer et al., 1966). The gradient of the irradiation line is lower, indicating a longer lag phase (Fig. 4). This suggests that the irradiation process led to sub-lethal injury. It can therefore be said that the observed lag phase extension is due to a decrease in cell number and to the repair of injured cells. The reasons for the response of the organism to the combined treatments is not clear, as there is little literature regarding the effects of alkaline conditions on lactic acid bacteria. The exponential culture may undergo an 'alkaline shock', i.e. an increase in hydroxyl ion (OH-)

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L. curvatus,

pH

84

L.M. Fielding, P.E. Cook, A.S. Grandison

concentration, when it is resuspended in modified broth at pH 7.0 and 7-5, or in standard broth (pH 6.1). The irradiation process itself also leads to an increase in the O H - concentration, as this ion is a radiolysis product of water. When the pH is modified prior to irradiation, to pH 7-0 and 7.5, and then irradiated, the O H - concentration is increased significantly. It has been suggested that irradiation affects cell membrane structure and permeability (Urbain, 1986. p. 84). This would allow influx of the O H ions into the cell cytoplasm and disrupt the pH homeostasis mechanism, a process requiring energy. The D N A repair mechanism also consumes energy and therefore the combined treatment of increased pH followed by irradiation may place significant stress on the cells. The survival and recovery of the organism after pH modification and irradiation may be dose dependent, as the effect was observed after a dose of 1-05 kGy (Fig. 6), but not after 0-35 kGy. This may relate to the effect of irradiation on cell membrane properties or less production of O H - ions by radiolysis of water. A possible reason why this does not occur when the pH is modified after irradiation is the speed at which DNA repair enzymes operate. The majority of radiation-damaged D N A is repaired very quickly after irradiation (Josephson & Peterson, 1983). The stress of DNA repair is therefore removed before the pH is raised significantly. It is not understood why there was little growth at pH 7.0 after irradiation. The evidence also suggests that the observed effect is dependent on the O H - concentration. Irradiation at pH 7-5 led to a greater decrease in number than irradiation at pH 7.0.

ACKNOWLEDGEMENTS We would like to thank Unilever Research, Colworth House, Bedford, for partial funding of this work. Particular thanks go to Professor Grahame Gould and Dr Ivar Assinder. We thank also Dr Vivian Dillon of the Department of Food Science and Technology, Reading University, for her help throughout this project.

REFERENCES Anon. (1991). FAO/IAEA Research Co-ordination Meeting on Irradiation in Combination with other Processes for Improving Food Quality, Strasbourg, France, 8-12th April IAEA, Vienna, pp. 5-8.

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Brock, T.D., Smith, D.W. & Madigan, M.T. (1970) Biology of Micro-Organisms, 4th edn. Prentice-Hall, Englewood Cliffs, NJ, p. 736. Dyer, J.K., Anderson, A.W. & Dutiyabodhi, P. (1966). Radiation survival of food pathogens in complex media. Applied Microbiology, 14, 92-97. Gould, G.W. (ed.) (1989). Mechanisms of Action of Food Preservation Procedures. Elsevier, Amsterdam, p. 401. Hastings, J.W., Holzapfel, W.H. & Niemand, J.G. (1986). Radiation resistance of lactobacilli isolated from radurised meat relative to growth and environment. Applied and Environmental Microbiology, 52, 898-901. Jay, J.M. (1978). Modern Food Microbiology, 2nd edn. Van Nostrand, London, p. 30. Josephson, E.S. & Peterson, M.S. (1982) Preservation of Food by Ionising Radiation, Iiol.1, CRC Press, Boca Raton, FL, p. 6. Josephson, E.S. & Peterson, M.S. (1983) Preservation of Food by lonising Radiation, Vol.ll, CRC Press, Boca Raton, FL, p. 167. Patterson, M.F. (1988). Sensitivity of bacteria to irradiation on poultry meat under various atmospheres. Letters in Applied Microbiology, 7, 55-58. Urbain, W.M. (1986). Food Irradiation. Academic Press, London, pp. 84, 118, 257.