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The effect of electron beam irradiation, combined with acetic acid, on the survival and recovery of Escherichia coli and Lactobacillus curvatus
lntemationd Joumal ofFoodl#ia&iology ELSEVIER
International Journal of Food Microbiology 35 (1997) 259-265
The effect of electron beam irradiation, combined with acetic acid, on the survival and recovery of Escherichia coli and Lactobacillus
curvatus
Louise M. Fielding *, Paul E. Cook, Alistair S. Grandison Department
of Food
Science
and Technology,
of Reading,
The Unicersity
Received 23 July 1996; received in revised form 23 December
Whiteknights,
1996; accepted
Reading
RG6 6A P, UK
30 December
1996
Abstract The preservation of food by ionising radiation may lead to undesirable sensory changes within the food. These changes can be reduced by combining irradiation with other treatments, for example the addition of organic acids. Late exponential phase cultures of Eschevichiu coli and Lactobacillus curcatus were irradiated, in a liquid medium, at doses of O-l.8 kilograys (kGy), in the presence of acetic acid (OP2%) at pH 4.6. A synergistic effect occurred when E. coli was irradiated in the presence of acetic acid (0.02- 1 .O’G) at all doses used (0.14% 1.1 kGy). There is evidence to suggest that membrane disruption occurred in the cells as a result of the combined treatments and this may account, to some extent, for the synergism observed. The addition of acetic acid up to a concentration of 2.0% had no effect upon the radiation survival or upon the subsequent growth of L. curuatus. 0 1997 Elsevier Science B.V. Keywords:
Food
irradiation;
Combination
treatments;
Organic
1. Introduction Two of the primary tion
are to eliminate
number of spoilage organisms present. The process can, however, lead to adverse sensory changes in the food. The severity of radiation processing may be reduced by combination with other treatments, thus ensuring the microbiological quality of the food without compromising its sensory properties (Urbain, 1986). In addition the cost of the process may be reduced and consumer acceptability may be improved as some harmful additives may be replaced or reduced by the concomitant use of other processes (Earnshaw, 1990).
objectives of food irradiapathogens and to reduce the
* Corresponding author. Present address: School of Food and Consumer Science, Faculty of Business, Leisure and Food, University of Wales Institute, Cardiff, Colchester Avenue Campus, Cardiff, CF3 7XR. UK. Tel.: +44 1222 506456; fax: + 44 1222 506941; e-mail: [email protected] 0168-I 605!97/$17.00
0 1997 Elsevier
PIISO168-1605(97)01251-8
Science B.V. All rights
acids
reserved
Low pH has long been used as a method of food preservation and, for a number of foods, is a natural defence mechanism (Brown and Mayes, 1980). Fruits are protected against bacterial invasion by their inherent acidity and organic acid content. Other foods, such as yoghurt and cheese, owe their acidity to the action of micro-organisms during fermentation processes (Booth and Kroll, 1989; Jay, 1992). The antimicrobial effects of organic acids are due to a combination of the hydrogen ion concentration (lowering the pH) and the fact that the undissociated molecule may freely permeate the cell membrane. Combination treatments, also known as hurdle technology, rely on the fact that the sensitivity of organisms to stresses imposed on them can be cumulative. Consequently, when a number of different preservative treatments are applied together, at subinhibitory levels, the overall effect may be to cause inhibition (Ray. 1992). The aim of this work was to determine the effects of acetic acid, in combination with electron beam irradiation, on the survival and recovery of Esclwrichia coli and Lactohucillus curvutus.
2. Materials
and methods
2. I. Organisms and nwdiu Late exponential phase cultures of E. coli (NCIMB 9270) and L. curvutus (NCFB 1041) were used. Details of the media used and method of preparation have been reported previously (Fielding et al., 1994a,b). Following the treatments outlined below, the numbers of survivors were enumerated on duplicate pour plates. Dilutions were prepared in buffered peptone water (BPW; Difco 1810-l 7-9) and the agars used were yeast glucose broth for E. coli and MRS broth for L. curcatus, both solidified with 12 g/l of agar (Oxoid L13). All plates were incubated at either 37°C for 24 h (E. co/i) or 30°C for 48 h (L. curwtw) prior to enumeration of colonies.
2.2. Tk
radiution source
Samples were irradiated using a Van de Graaff electron beam accelerator (model AK, Cambridge, USA) which has an energy range of 0.5 2.0 MeV and a current range of O--50 /iA. The sample was placed on a conveyor which passed beneath the scanning horn, at a fixed speed, for a fixed period of time. The dose was varied by altering the beam current up to a maximum of 30 /IA (a higher current could not be used, without considerable shielding, due to gamma scatter). The dose absorbed was measured using optichromic dosemeters (FWT-70-83, Far-West Technology, Goleta, CA) and a spectrophotometer (FWT-98 opti-chromic reader). The dose rate varied from 0 to 0.75 kGy/s. 2.3. Luh.sy.stem.s bioscreen The Bioscreen (Labsystems, Basingstoke. UK) is an automated microbial growth analyser. The absorbance of multiple samples is measured by vertical light photometry over a specified period of time. The optical density of a bacterial suspension is directly proportional to the number of cells present and a growth, or turbidometric, curve may, therefore, be obtained. Operating details have been described by Fielding et al. (1994a). The parameter used to analyse the Bioscreen results was the time taken for a culture to reach a given absorbance value at 600 nm. As the number of cells present in a sample at time zero decreases, the time taken for that culture to reach the predetermined absorbance increases. This is because the equipment will not detect an increase in absorbance until the cell number reaches approximately 10”:‘ml. It is possible to quantify injury using this method as sublethally stressed cells may operate repair mechanisms which will result in 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. A calibration of the Bioscreen was needed to compare the absorbance values obtained with the actual number of cells present. A 24 hour subculture of each organism was centrifuged at 6000 x g for 10 min (IEC Centra-4X Bench Top Cen-
L.M.
Fielding et al. /International
Journal of‘ Food Microbiology
trifuge, Dunstable, UK) and the pellet was resuspended in 10 ml of growth medium (YGB or MRS-B) which had been modified to pH 4.6 using 0.1 mol/l HCl. A series of twofold dilutions were subsequently prepared in growth medium (pH 4.6). A control was also prepared which consisted of sterile broth. This type of control was used in all Bioscreen analyses. Four hundred microlitres of each dilution were pipetted, in triplicate, into a Bioscreen plate which was then incubated at 37°C (E. coli) or 30°C (L. curuatus). Plate counts were also carried out. From the Bioscreen, the time taken for each dilution to reach a given absorbance value was determined. These values were plotted against log colony forming units per ml (log cfu/ml) to give a calibration curve. Assuming that a linear relationship existed, the coefficients of the equation (Eq. (1)) for linear regression were calculated for each organism. Y=A+Bx
(1)
where y is log cfu/ml, x is the time taken to reach the predetermined absorbance value (h), A is the intercept of the line on the Y axis and B is the gradient of the line. 2.4. The ejjixt
of’ acetic
acid
The effect of modified pH and irradiation as both single and combined stresses has been reported previously (Fielding et al., 1994a,b). Acetic (ethanoic) acid was used in this study at a pH of 4.6. A 10% solution of the acid was added to double strength growth medium to the desired concentration. The pH was then modified to 4.6 using 0.1 mol/l HCl or 0.1 mol/l NaOH. The volume of the broth was then made up to 10 ml using sterile, high purity water. The late exponential phase culture was centrifuged at 6000 x g for 10 min and the pellet was resuspended in the acid and pH-modified medium. Duplicate samples were prepared. After incubation at 25°C for 1.5 h, a 10 ~ ’ dilution was made in a further 9 ml of acid and pH-modified broth. A Bioscreen plate was set up and pour plates were prepared. The remainder of the inoculated modified broth was retained and incubated at 25°C for a further 72 h after which time 5 ml of the sample was
35 (1997) 259-265
261
removed and centrifuged. The cells were resuspended in BPW (to remove traces of acid and to neutralise the pH) and a ten-fold dilution was prepared in standard medium. A Bioscreen plate and pour plates were prepared. 2.5. The ejfect of acetic acid combined irradiation
with
The acid and pH-modified broth was prepared as described above. The centrifuged cells were resuspended in broth and transferred to a ‘celcult’ 6-well flat bottomed plate (Sterilin 33F06L). After incubation at 25°C for 1.5 h the cells were irradiated, at ambient temperature, at doses of between 0 and 2.0 kGy. Duplicate samples were prepared. Approximately 20 min after irradiation a 10 ~ ’ dilution was made in a further 9 ml of acid and pH-modified broth. A Bioscreen plate and pour plates were prepared. Each experiment was duplicated. 2.6. Determinution
of membrane
damage
Organic acid and ionising radiation both have the ability to disrupt the membranes of bacterial cells. Two methods were used to determine the extent of membrane damage in E. coli. Firstly, the sensitivity of the treated cells to the presence of an antibiotic in the plating medium was determined. The antibiotic used was Actinomycin-D (Act-D) which is a peptide and has the ability to bind to DNA and prevent transcription of DNA to RNA. The production of enzymes and proteins required by the cell is thus inhibited (Betzel et al., 1993). Act-D does not have the ability to permeate the intact membranes of bacterial cells, but only those which have suffered damage to the membranes (Davies, personal communication). The method of enumeration of viable cells was identical to that described above. Two sets of agar were prepared, one which contained no Act-D and one to which 2 ppm of Act-d had been added after tempering. This concentration had been previously determined as the lowest which gave a reduction in the number of viable cells recovered after irradiationa treatment known to damage cell membranes. Plates were prepared for the samples with each of
L.M.
262
Fielding et 01. 1Intrrnutionul
Journul
the agars and incubated as before. Those plates containing between 30-300 colonies were counted and any difference in the number of viable cells recovered from the two agars was noted. The second method used was measurement of the absorbance of the broth in which the cells were treated. This should provide information on the leakage of cytoplasmic markers such as potassium ions, amino acids and ribonucleotides. The treated cells were removed from the ccl-cult plates and centrifuged at 5000 rev/min for 10 min. The resulting supernatant was poured off and placed, aseptically, into a sterile universal bottle. This supernatant was stored at - 80°C until required. The broth was defrosted at room temperature and was diluted in deionised water (0.1 ml broth in 2 ml water) to bring the absorbance within the range of the equipment (Perkin Elmer UVjVis. Spectrophotometer, Lambda 3B). The wavelength was set at 260 nm and deionised water was used as a reference. The samples were placed in UV disposable cuvettes and the absorbance of triplicate samples was measured. The absorbance of a broth, in which E. coli had been placed at pH 7.0 for 1.5 h before centrifuging, was also measured to act as a control.
of Food
Mic~rohiolog~:?’ 35 (1997) 259
265
organism, however, showed a decrease in cfu of approximately 4 log cycles after 72 h incubation in 1% acetic acid (results not included). The Bioscreen results demonstrate that incubation in 0.12% acid led to growth inhibition of E. coli (Fig. 1). Fig. 2 shows good recovery of the organism after 72 h incubation followed by inoculation in standard growth medium, up to concentrations of 0.2% acetic acid. Analysis of covariance showed that there is a significant difference in the gradients of the lines obtained after incubation of E. coli in acetic acid for 72 h. The plate count results for L. curccttus show that this organism is capable of survival and growth in concentrations of acetic acid up to 1%. The Bioscreen results (Fig. 3) show minimal growth of L. curcatus in 1% acetic acid. The growth rate at all concentrations was reduced and no growth was observed in 1.5% acid. Analysis of covariance showed a significant extension of the lag phase of L. cvrcutus when it was incubated in acetic acid. After 72 h incubation, followed by inoculation in standard MRS broth, recovery of the organisms at all concentrations of acetic acid was demonstrated (Fig. 4). A significant increase
3. Results 3.1. Cdibrution ?If’ the Bioscreen at pH 4.6 The time taken for the organisms to reach an absorbance of 0.50 (E. coli) or 0.602 (L. curwtu~) was plotted against log cfu/ml and the coefficients of the equations were determined (Eq. (2) E. coli; Eq. (3) L. curvutus) by linear regression and are given below. E. coli,
9.125 - 0.599~
L. curvatus,
P = 0.998
7.748 - 0.245~
r = 0.993
(2) (3) 0
0
500
1000 TIME
1500
2000
2500
300
(MINUTES)
4. The effect of acetic acid alone The pour plate results show that the presence of acetic acid at pH 4.6 had little effect on the survival of E. coli after incubation for 1.5 h. This
Fig. I. The effect of acetic acid (O-~0.12%) at pH 4.6 on the growth of E. co/r after 1.5 h incubation, measured in the I. 0%; x, O.O05’!k ,7. Bioscreen; acetic acid concentration: 0.02’%,; #, 0.04%: 0. 0.06%; +. O.OS’%,: I:, 0.1%;( 0.12%;_ control.
L.M.
Fielding et al. /International
1 i-
Journal qf Food Microbiology
I
35 (1997) 259-265
measured by the Bioscreen, the presence of acetic acid. 5.1. Determination
0
0
300
600
900
TIME
(MINUTES)
1200
150
Fig. 2. The effect of 72 h incubation in acetic acid (O-0.2%) at pH 4.6 followed by inoculation in standard growth medium, on the growth of E. coli measured using the Bioscreen. Acetic acid concentration: 3, On/;,;x, 0.01%; V, 0.02%; #, 0.05%; 0, 0.075%; +, 0.1%; n, 0.15%; , 0.12%.
in lag phase with increasing acetic acid concentration was demonstrated by analysis of covariance.
263
was not affected
of membrane
by
damage
The extent of membrane damage was determined for the effect of acetic acid at 0 and 0.53 kGy on E. coli. This combination was chosen as it is one that exhibited a synergistic effect. It was found that the addition of Act-D to the plating medium gave no reduction in the number of viable cells recovered when either irradiation or acetic acid was used as a single treatment. When the two were combined,. however, at a radiation dose of 0.53 kGy there was a significant reduction in the number of colonies enumerated (P < 0.02). No analysis of membrane damage was carried out for L. curvutus as this organism did seem to be affected by the presence of acetic acid during irradiation. The absorbance results obtained (the average of three replicates-A,,) were examined using analysis of variance, in the form of a Student’s t-test (2-tailed) to determine the probability of the results being from the same population. The results showed that there was a significant difference
5. The effect of acetic acid combined with irradiation The effect of irradiation on E. coli, at all doses, was enhanced by the presence of acetic acid (0.02P1%), resulting in a marked reduction in numbers (Fig. 5). At all doses used and at concentrations of acetic acid above 0.05% the extent of this synergism seems to be independent of concentration (approximately 3 log cycles at 0.6 kGy). At 0.02% the effect was slightly reduced (approximately 2.5 log cycles at 0.6 kGy). Fig. 6 shows the growth of E. coli in 0.05% acid, after irradiation in 0.05% acetic acid. It can be seen that, after irradiation, the growth of the organism is retarded by the presence of the acid. The same effect was seen at all concentrations of acid above 0.05% but not at 0.02% No synergistic effect was seen when L. curvatus was irradiated at doses of up to 1.8 kGy in concentrations of acetic acid up to 2%. The growth of this organism following irradiation, as
0
500
1000 TIME
1500
2000
2500
300
(MINUTES)
Fig. 3. The effect of acetic acid (O-2.5%) at pH 4.6 on the growth of L. curcatus after 1.5 h incubation, measured in the Bioscreen. Acetic acid concentration: 0, 0%; x, 0.2%; V, 0.4%. #, 0.6%; 0, 0.8%; +, 1.0%; a, 1.5%; , 2.0%; , 2.5%.
264
L.M.
Fielding et al.
Interncrtiond
Journal of’ Food Miuohiology
3.5 (1997) 259-265
.s
.I
0
0
1000
500
TIME
1500
2000
2500
300
(MINUTES)
0 0
600
1200 TIME
Fig. 4. The effect of 72 h incubation in acetic acid (O-2.5%) at pH 4.6 followed by inoculation in standard growth medium, on the growth of L. cumztusmeasured using the Bioscreen. Acetic acid concentration: C, O’%,;x, 0.2%: :‘. 0.4%; # . 0.6%; 0, 0.8%; + . I .O’%>;a, I .5’S; , 2.0%; . 2.5%.
between the control broth (A,,, 1.056) and the broths in which E. coli had been treated with irradiation (A,,, 1.318; P < 0.05), acetic acid at
1800
2400
300
(MINUTES)
Fig. 6. The effect of irradiation (0 0.42 kGy) in the presence of 0.05% acetic acid on the subsequent growth of E. coli at pH 4.6, measured using the Bioscreen. Acetic acid concentration: dose (kGy): r. 0; X, 0.145; V, 0.42.
pH 4.6 (A,,., 1.256; P < 0.20) and a combination of the two (A;,,., 1.486; P < 0.01).
6. Discussion
.2
.4
.6 DOSE
.8
1
1.2
(kGy)
Fig. 5. The effect of irradiation (O- 1. I kGy) in the presence of acetic acid (O-l’%,) at pH 4.6 on the survival of E. co/i, measured using pour plates. Acetic acid concentration: Ll, O’%I; x, 0.02%: V, 0.05%: #, 0.1%; 0. 0.3%: +, 0.5%; n. 1.0%.
The reason for the response of E. mli to the presence of acetic acid during irradiation has at least three possibilities. The acid may sensitise the organism to irradiation by the disruption of metabolic functions, such as energy production or enzyme activity, or the increased energy demand of homeostasis placed on the cell may inhibit DNA repair mechanisms. Conversely, the radiation process may sensitise the organism to the presence of acetic acid after irradiation by injury to the cell membrane allowing uncontrolled influx of acid molecules. This is unlikely as the undissociated acetic acid has the ability to permeate the cell membrane freely. Another theory is that the effect of irradiation on the acetic acid molecule produces a lethal free radical which subsequently acts on the cell. If this were the case it may be said that the synergism observed is an indirect effect as neither the irradiation nor the acetic acid molecule itself is producing the effect.
L.M.
Fidding
et al. /International
Journal
Although the concentration of acetic acid during irradiation had little effect on the extent of the synergistic response, E. coli was more susceptible to higher concentrations of the acid in the growth medium after irradiation. This may be explained by the increased energy demands on the cell as a result of homeostatic mechanisms. The results from the measurement of membrane damage suggest that the combination of acetic acid with irradiation results in more damage to this organelle than the use of irradiation alone. L. curvatus did not give the same response as E. coli to the combined stresses of acetic acid during irradiation. This may be explained by the fact that the lactic acid bacteria are much more resistant to the effects of weak organic acids. If a lethal free radical is the cause of the response in E. coli it is possible that this radical would not have the same effect on L. cuuoutus as this organism is more tolerant to both acetic acid and irradiation. As stated by Booth “the synergism between low pH, weak acids and irradiation is not surprising but the demonstration is novel” (personal communication). The theories described above cannot fully explain the results obtained but a number of conclusions can, however, be drawn. It can be concluded that the use of organic acids in conjunction with irradiation has shown some potential for the destruction of pathogenic organisms in a model system. The application of these stresses in a real food system has not yet been investigated. Acetic acid does not seem to have any application in the control of lactic acid food spoilage bacteria. It is unlikely that investigation in a real food system would contradict this as the components present in food will protect the organism from the lethal effects of irradiation (Quinn et al., 1967). One important potential use arising from these results is the application of organisms of the genus Lactohacillus as competitive organisms to control pathogens. It has been shown that irradiation, at modified pH, in the presence of low concentrations (0.02%) of acetic acid acted synergistically on the destruction of E. coli. This com-
qf Food
Microhiolog~~
35 (1997) 259-265
265
did not have the same effect on L. It is possible, therefore, that irradiation of food, in conjunction with the above named treatments, could lead to the production of a safe commodity with a longer shelf-life. The food would spoil, as a result of the growth of lactic acid bacteria, before any surviving pathogens could grow to numbers sufficient to present a hazard. bination
curmtus.
References Betzel, C., Rachev, R., Dolashka. P. and Genov. N. (1993) Actinomycins as proteinase inhibitors. Biochim. Biophys. Acta Protein Struct. Mol. Enzymol. 1161 (I), 47-51. Booth, I.R. and Kroll, R.G. (1989) The preservation of food by low pH. In: G.W. Gould (editor), Mechanisms of Action of Food Preservation Procedures. Elsevier, London, pp. 119~160. Brown, M.H. and Mayes, T. (1980) The growth of microbes at low pH values. In: G.W. Gould and J.E.L. Corry (editors), Microbial Growth and Survival in Extremes of Environment SAB Technical Series No. 15. Academic Press, London, pp. 71-98. Earnshaw, R. (1990) Use of combination processes: their benefits. In: Irradiation and Combination Treatments. Conf. Proc.. IBC Technical Services Ltd., London, I-2 March. Fielding, L.M., Cook, P.E. and Grdndison, AS. (1994a) The effect of irradiation and pH on Lactohacihs wruafus. Int. Biodeterior. Biodegrad. 32. 75-85. Fielding. L.M.. Cook. P.E. and Grandison, A.S. (1994b) The effect of electron beam irradiation and modified pH on the survival and recovery of Escherictriu cdi. J. Appl. Bacteriol. 76. 412-416. Jay, J.M. (1992) Modern Food Microbiology, 4th edition. Van Nostrand. London, pp. 371. Quinn, D.J., Anderson, A.W. and Dyer, J.F. (1967) The inactivation of infection and intoxification micro-organisms by irradiation in seafood. In: Microbiological Problems in Food Preservation by Irradiation. Proc. Panel, Vienna, 27 June-l July 1966, organised by the Joint FAO! IAEA Division of Atomic Energy in Food and Agriculture. IAEA. Vienna, pp. I-13. Ray, B. (1992) The need for food biopreservation. In: B. Ray and M. Daeschel (editors), Food Biopreservatives of Microbial Origin. CRC Press, Boca Raton, FL. pp. I-23. Urbain. W.M. (1986) Food Irradiation. Academic Press, New York.
International Journal of Food Microbiology 35 (1997) 259-265
The effect of electron beam irradiation, combined with acetic acid, on the survival and recovery of Escherichia coli and Lactobacillus
curvatus
Louise M. Fielding *, Paul E. Cook, Alistair S. Grandison Department
of Food
Science
and Technology,
of Reading,
The Unicersity
Received 23 July 1996; received in revised form 23 December
Whiteknights,
1996; accepted
Reading
RG6 6A P, UK
30 December
1996
Abstract The preservation of food by ionising radiation may lead to undesirable sensory changes within the food. These changes can be reduced by combining irradiation with other treatments, for example the addition of organic acids. Late exponential phase cultures of Eschevichiu coli and Lactobacillus curcatus were irradiated, in a liquid medium, at doses of O-l.8 kilograys (kGy), in the presence of acetic acid (OP2%) at pH 4.6. A synergistic effect occurred when E. coli was irradiated in the presence of acetic acid (0.02- 1 .O’G) at all doses used (0.14% 1.1 kGy). There is evidence to suggest that membrane disruption occurred in the cells as a result of the combined treatments and this may account, to some extent, for the synergism observed. The addition of acetic acid up to a concentration of 2.0% had no effect upon the radiation survival or upon the subsequent growth of L. curuatus. 0 1997 Elsevier Science B.V. Keywords:
Food
irradiation;
Combination
treatments;
Organic
1. Introduction Two of the primary tion
are to eliminate
number of spoilage organisms present. The process can, however, lead to adverse sensory changes in the food. The severity of radiation processing may be reduced by combination with other treatments, thus ensuring the microbiological quality of the food without compromising its sensory properties (Urbain, 1986). In addition the cost of the process may be reduced and consumer acceptability may be improved as some harmful additives may be replaced or reduced by the concomitant use of other processes (Earnshaw, 1990).
objectives of food irradiapathogens and to reduce the
* Corresponding author. Present address: School of Food and Consumer Science, Faculty of Business, Leisure and Food, University of Wales Institute, Cardiff, Colchester Avenue Campus, Cardiff, CF3 7XR. UK. Tel.: +44 1222 506456; fax: + 44 1222 506941; e-mail: [email protected] 0168-I 605!97/$17.00
0 1997 Elsevier
PIISO168-1605(97)01251-8
Science B.V. All rights
acids
reserved
Low pH has long been used as a method of food preservation and, for a number of foods, is a natural defence mechanism (Brown and Mayes, 1980). Fruits are protected against bacterial invasion by their inherent acidity and organic acid content. Other foods, such as yoghurt and cheese, owe their acidity to the action of micro-organisms during fermentation processes (Booth and Kroll, 1989; Jay, 1992). The antimicrobial effects of organic acids are due to a combination of the hydrogen ion concentration (lowering the pH) and the fact that the undissociated molecule may freely permeate the cell membrane. Combination treatments, also known as hurdle technology, rely on the fact that the sensitivity of organisms to stresses imposed on them can be cumulative. Consequently, when a number of different preservative treatments are applied together, at subinhibitory levels, the overall effect may be to cause inhibition (Ray. 1992). The aim of this work was to determine the effects of acetic acid, in combination with electron beam irradiation, on the survival and recovery of Esclwrichia coli and Lactohucillus curvutus.
2. Materials
and methods
2. I. Organisms and nwdiu Late exponential phase cultures of E. coli (NCIMB 9270) and L. curvutus (NCFB 1041) were used. Details of the media used and method of preparation have been reported previously (Fielding et al., 1994a,b). Following the treatments outlined below, the numbers of survivors were enumerated on duplicate pour plates. Dilutions were prepared in buffered peptone water (BPW; Difco 1810-l 7-9) and the agars used were yeast glucose broth for E. coli and MRS broth for L. curcatus, both solidified with 12 g/l of agar (Oxoid L13). All plates were incubated at either 37°C for 24 h (E. co/i) or 30°C for 48 h (L. curwtw) prior to enumeration of colonies.
2.2. Tk
radiution source
Samples were irradiated using a Van de Graaff electron beam accelerator (model AK, Cambridge, USA) which has an energy range of 0.5 2.0 MeV and a current range of O--50 /iA. The sample was placed on a conveyor which passed beneath the scanning horn, at a fixed speed, for a fixed period of time. The dose was varied by altering the beam current up to a maximum of 30 /IA (a higher current could not be used, without considerable shielding, due to gamma scatter). The dose absorbed was measured using optichromic dosemeters (FWT-70-83, Far-West Technology, Goleta, CA) and a spectrophotometer (FWT-98 opti-chromic reader). The dose rate varied from 0 to 0.75 kGy/s. 2.3. Luh.sy.stem.s bioscreen The Bioscreen (Labsystems, Basingstoke. UK) is an automated microbial growth analyser. The absorbance of multiple samples is measured by vertical light photometry over a specified period of time. The optical density of a bacterial suspension is directly proportional to the number of cells present and a growth, or turbidometric, curve may, therefore, be obtained. Operating details have been described by Fielding et al. (1994a). The parameter used to analyse the Bioscreen results was the time taken for a culture to reach a given absorbance value at 600 nm. As the number of cells present in a sample at time zero decreases, the time taken for that culture to reach the predetermined absorbance increases. This is because the equipment will not detect an increase in absorbance until the cell number reaches approximately 10”:‘ml. It is possible to quantify injury using this method as sublethally stressed cells may operate repair mechanisms which will result in 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. A calibration of the Bioscreen was needed to compare the absorbance values obtained with the actual number of cells present. A 24 hour subculture of each organism was centrifuged at 6000 x g for 10 min (IEC Centra-4X Bench Top Cen-
L.M.
Fielding et al. /International
Journal of‘ Food Microbiology
trifuge, Dunstable, UK) and the pellet was resuspended in 10 ml of growth medium (YGB or MRS-B) which had been modified to pH 4.6 using 0.1 mol/l HCl. A series of twofold dilutions were subsequently prepared in growth medium (pH 4.6). A control was also prepared which consisted of sterile broth. This type of control was used in all Bioscreen analyses. Four hundred microlitres of each dilution were pipetted, in triplicate, into a Bioscreen plate which was then incubated at 37°C (E. coli) or 30°C (L. curuatus). Plate counts were also carried out. From the Bioscreen, the time taken for each dilution to reach a given absorbance value was determined. These values were plotted against log colony forming units per ml (log cfu/ml) to give a calibration curve. Assuming that a linear relationship existed, the coefficients of the equation (Eq. (1)) for linear regression were calculated for each organism. Y=A+Bx
(1)
where y is log cfu/ml, x is the time taken to reach the predetermined absorbance value (h), A is the intercept of the line on the Y axis and B is the gradient of the line. 2.4. The ejjixt
of’ acetic
acid
The effect of modified pH and irradiation as both single and combined stresses has been reported previously (Fielding et al., 1994a,b). Acetic (ethanoic) acid was used in this study at a pH of 4.6. A 10% solution of the acid was added to double strength growth medium to the desired concentration. The pH was then modified to 4.6 using 0.1 mol/l HCl or 0.1 mol/l NaOH. The volume of the broth was then made up to 10 ml using sterile, high purity water. The late exponential phase culture was centrifuged at 6000 x g for 10 min and the pellet was resuspended in the acid and pH-modified medium. Duplicate samples were prepared. After incubation at 25°C for 1.5 h, a 10 ~ ’ dilution was made in a further 9 ml of acid and pH-modified broth. A Bioscreen plate was set up and pour plates were prepared. The remainder of the inoculated modified broth was retained and incubated at 25°C for a further 72 h after which time 5 ml of the sample was
35 (1997) 259-265
261
removed and centrifuged. The cells were resuspended in BPW (to remove traces of acid and to neutralise the pH) and a ten-fold dilution was prepared in standard medium. A Bioscreen plate and pour plates were prepared. 2.5. The ejfect of acetic acid combined irradiation
with
The acid and pH-modified broth was prepared as described above. The centrifuged cells were resuspended in broth and transferred to a ‘celcult’ 6-well flat bottomed plate (Sterilin 33F06L). After incubation at 25°C for 1.5 h the cells were irradiated, at ambient temperature, at doses of between 0 and 2.0 kGy. Duplicate samples were prepared. Approximately 20 min after irradiation a 10 ~ ’ dilution was made in a further 9 ml of acid and pH-modified broth. A Bioscreen plate and pour plates were prepared. Each experiment was duplicated. 2.6. Determinution
of membrane
damage
Organic acid and ionising radiation both have the ability to disrupt the membranes of bacterial cells. Two methods were used to determine the extent of membrane damage in E. coli. Firstly, the sensitivity of the treated cells to the presence of an antibiotic in the plating medium was determined. The antibiotic used was Actinomycin-D (Act-D) which is a peptide and has the ability to bind to DNA and prevent transcription of DNA to RNA. The production of enzymes and proteins required by the cell is thus inhibited (Betzel et al., 1993). Act-D does not have the ability to permeate the intact membranes of bacterial cells, but only those which have suffered damage to the membranes (Davies, personal communication). The method of enumeration of viable cells was identical to that described above. Two sets of agar were prepared, one which contained no Act-D and one to which 2 ppm of Act-d had been added after tempering. This concentration had been previously determined as the lowest which gave a reduction in the number of viable cells recovered after irradiationa treatment known to damage cell membranes. Plates were prepared for the samples with each of
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Journul
the agars and incubated as before. Those plates containing between 30-300 colonies were counted and any difference in the number of viable cells recovered from the two agars was noted. The second method used was measurement of the absorbance of the broth in which the cells were treated. This should provide information on the leakage of cytoplasmic markers such as potassium ions, amino acids and ribonucleotides. The treated cells were removed from the ccl-cult plates and centrifuged at 5000 rev/min for 10 min. The resulting supernatant was poured off and placed, aseptically, into a sterile universal bottle. This supernatant was stored at - 80°C until required. The broth was defrosted at room temperature and was diluted in deionised water (0.1 ml broth in 2 ml water) to bring the absorbance within the range of the equipment (Perkin Elmer UVjVis. Spectrophotometer, Lambda 3B). The wavelength was set at 260 nm and deionised water was used as a reference. The samples were placed in UV disposable cuvettes and the absorbance of triplicate samples was measured. The absorbance of a broth, in which E. coli had been placed at pH 7.0 for 1.5 h before centrifuging, was also measured to act as a control.
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organism, however, showed a decrease in cfu of approximately 4 log cycles after 72 h incubation in 1% acetic acid (results not included). The Bioscreen results demonstrate that incubation in 0.12% acid led to growth inhibition of E. coli (Fig. 1). Fig. 2 shows good recovery of the organism after 72 h incubation followed by inoculation in standard growth medium, up to concentrations of 0.2% acetic acid. Analysis of covariance showed that there is a significant difference in the gradients of the lines obtained after incubation of E. coli in acetic acid for 72 h. The plate count results for L. curccttus show that this organism is capable of survival and growth in concentrations of acetic acid up to 1%. The Bioscreen results (Fig. 3) show minimal growth of L. curcatus in 1% acetic acid. The growth rate at all concentrations was reduced and no growth was observed in 1.5% acid. Analysis of covariance showed a significant extension of the lag phase of L. cvrcutus when it was incubated in acetic acid. After 72 h incubation, followed by inoculation in standard MRS broth, recovery of the organisms at all concentrations of acetic acid was demonstrated (Fig. 4). A significant increase
3. Results 3.1. Cdibrution ?If’ the Bioscreen at pH 4.6 The time taken for the organisms to reach an absorbance of 0.50 (E. coli) or 0.602 (L. curwtu~) was plotted against log cfu/ml and the coefficients of the equations were determined (Eq. (2) E. coli; Eq. (3) L. curvutus) by linear regression and are given below. E. coli,
9.125 - 0.599~
L. curvatus,
P = 0.998
7.748 - 0.245~
r = 0.993
(2) (3) 0
0
500
1000 TIME
1500
2000
2500
300
(MINUTES)
4. The effect of acetic acid alone The pour plate results show that the presence of acetic acid at pH 4.6 had little effect on the survival of E. coli after incubation for 1.5 h. This
Fig. I. The effect of acetic acid (O-~0.12%) at pH 4.6 on the growth of E. co/r after 1.5 h incubation, measured in the I. 0%; x, O.O05’!k ,7. Bioscreen; acetic acid concentration: 0.02’%,; #, 0.04%: 0. 0.06%; +. O.OS’%,: I:, 0.1%;( 0.12%;_ control.
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measured by the Bioscreen, the presence of acetic acid. 5.1. Determination
0
0
300
600
900
TIME
(MINUTES)
1200
150
Fig. 2. The effect of 72 h incubation in acetic acid (O-0.2%) at pH 4.6 followed by inoculation in standard growth medium, on the growth of E. coli measured using the Bioscreen. Acetic acid concentration: 3, On/;,;x, 0.01%; V, 0.02%; #, 0.05%; 0, 0.075%; +, 0.1%; n, 0.15%; , 0.12%.
in lag phase with increasing acetic acid concentration was demonstrated by analysis of covariance.
263
was not affected
of membrane
by
damage
The extent of membrane damage was determined for the effect of acetic acid at 0 and 0.53 kGy on E. coli. This combination was chosen as it is one that exhibited a synergistic effect. It was found that the addition of Act-D to the plating medium gave no reduction in the number of viable cells recovered when either irradiation or acetic acid was used as a single treatment. When the two were combined,. however, at a radiation dose of 0.53 kGy there was a significant reduction in the number of colonies enumerated (P < 0.02). No analysis of membrane damage was carried out for L. curvutus as this organism did seem to be affected by the presence of acetic acid during irradiation. The absorbance results obtained (the average of three replicates-A,,) were examined using analysis of variance, in the form of a Student’s t-test (2-tailed) to determine the probability of the results being from the same population. The results showed that there was a significant difference
5. The effect of acetic acid combined with irradiation The effect of irradiation on E. coli, at all doses, was enhanced by the presence of acetic acid (0.02P1%), resulting in a marked reduction in numbers (Fig. 5). At all doses used and at concentrations of acetic acid above 0.05% the extent of this synergism seems to be independent of concentration (approximately 3 log cycles at 0.6 kGy). At 0.02% the effect was slightly reduced (approximately 2.5 log cycles at 0.6 kGy). Fig. 6 shows the growth of E. coli in 0.05% acid, after irradiation in 0.05% acetic acid. It can be seen that, after irradiation, the growth of the organism is retarded by the presence of the acid. The same effect was seen at all concentrations of acid above 0.05% but not at 0.02% No synergistic effect was seen when L. curvatus was irradiated at doses of up to 1.8 kGy in concentrations of acetic acid up to 2%. The growth of this organism following irradiation, as
0
500
1000 TIME
1500
2000
2500
300
(MINUTES)
Fig. 3. The effect of acetic acid (O-2.5%) at pH 4.6 on the growth of L. curcatus after 1.5 h incubation, measured in the Bioscreen. Acetic acid concentration: 0, 0%; x, 0.2%; V, 0.4%. #, 0.6%; 0, 0.8%; +, 1.0%; a, 1.5%; , 2.0%; , 2.5%.
264
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.s
.I
0
0
1000
500
TIME
1500
2000
2500
300
(MINUTES)
0 0
600
1200 TIME
Fig. 4. The effect of 72 h incubation in acetic acid (O-2.5%) at pH 4.6 followed by inoculation in standard growth medium, on the growth of L. cumztusmeasured using the Bioscreen. Acetic acid concentration: C, O’%,;x, 0.2%: :‘. 0.4%; # . 0.6%; 0, 0.8%; + . I .O’%>;a, I .5’S; , 2.0%; . 2.5%.
between the control broth (A,,, 1.056) and the broths in which E. coli had been treated with irradiation (A,,, 1.318; P < 0.05), acetic acid at
1800
2400
300
(MINUTES)
Fig. 6. The effect of irradiation (0 0.42 kGy) in the presence of 0.05% acetic acid on the subsequent growth of E. coli at pH 4.6, measured using the Bioscreen. Acetic acid concentration: dose (kGy): r. 0; X, 0.145; V, 0.42.
pH 4.6 (A,,., 1.256; P < 0.20) and a combination of the two (A;,,., 1.486; P < 0.01).
6. Discussion
.2
.4
.6 DOSE
.8
1
1.2
(kGy)
Fig. 5. The effect of irradiation (O- 1. I kGy) in the presence of acetic acid (O-l’%,) at pH 4.6 on the survival of E. co/i, measured using pour plates. Acetic acid concentration: Ll, O’%I; x, 0.02%: V, 0.05%: #, 0.1%; 0. 0.3%: +, 0.5%; n. 1.0%.
The reason for the response of E. mli to the presence of acetic acid during irradiation has at least three possibilities. The acid may sensitise the organism to irradiation by the disruption of metabolic functions, such as energy production or enzyme activity, or the increased energy demand of homeostasis placed on the cell may inhibit DNA repair mechanisms. Conversely, the radiation process may sensitise the organism to the presence of acetic acid after irradiation by injury to the cell membrane allowing uncontrolled influx of acid molecules. This is unlikely as the undissociated acetic acid has the ability to permeate the cell membrane freely. Another theory is that the effect of irradiation on the acetic acid molecule produces a lethal free radical which subsequently acts on the cell. If this were the case it may be said that the synergism observed is an indirect effect as neither the irradiation nor the acetic acid molecule itself is producing the effect.
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et al. /International
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Although the concentration of acetic acid during irradiation had little effect on the extent of the synergistic response, E. coli was more susceptible to higher concentrations of the acid in the growth medium after irradiation. This may be explained by the increased energy demands on the cell as a result of homeostatic mechanisms. The results from the measurement of membrane damage suggest that the combination of acetic acid with irradiation results in more damage to this organelle than the use of irradiation alone. L. curvatus did not give the same response as E. coli to the combined stresses of acetic acid during irradiation. This may be explained by the fact that the lactic acid bacteria are much more resistant to the effects of weak organic acids. If a lethal free radical is the cause of the response in E. coli it is possible that this radical would not have the same effect on L. cuuoutus as this organism is more tolerant to both acetic acid and irradiation. As stated by Booth “the synergism between low pH, weak acids and irradiation is not surprising but the demonstration is novel” (personal communication). The theories described above cannot fully explain the results obtained but a number of conclusions can, however, be drawn. It can be concluded that the use of organic acids in conjunction with irradiation has shown some potential for the destruction of pathogenic organisms in a model system. The application of these stresses in a real food system has not yet been investigated. Acetic acid does not seem to have any application in the control of lactic acid food spoilage bacteria. It is unlikely that investigation in a real food system would contradict this as the components present in food will protect the organism from the lethal effects of irradiation (Quinn et al., 1967). One important potential use arising from these results is the application of organisms of the genus Lactohacillus as competitive organisms to control pathogens. It has been shown that irradiation, at modified pH, in the presence of low concentrations (0.02%) of acetic acid acted synergistically on the destruction of E. coli. This com-
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did not have the same effect on L. It is possible, therefore, that irradiation of food, in conjunction with the above named treatments, could lead to the production of a safe commodity with a longer shelf-life. The food would spoil, as a result of the growth of lactic acid bacteria, before any surviving pathogens could grow to numbers sufficient to present a hazard. bination
curmtus.
References Betzel, C., Rachev, R., Dolashka. P. and Genov. N. (1993) Actinomycins as proteinase inhibitors. Biochim. Biophys. Acta Protein Struct. Mol. Enzymol. 1161 (I), 47-51. Booth, I.R. and Kroll, R.G. (1989) The preservation of food by low pH. In: G.W. Gould (editor), Mechanisms of Action of Food Preservation Procedures. Elsevier, London, pp. 119~160. Brown, M.H. and Mayes, T. (1980) The growth of microbes at low pH values. In: G.W. Gould and J.E.L. Corry (editors), Microbial Growth and Survival in Extremes of Environment SAB Technical Series No. 15. Academic Press, London, pp. 71-98. Earnshaw, R. (1990) Use of combination processes: their benefits. In: Irradiation and Combination Treatments. Conf. Proc.. IBC Technical Services Ltd., London, I-2 March. Fielding, L.M., Cook, P.E. and Grdndison, AS. (1994a) The effect of irradiation and pH on Lactohacihs wruafus. Int. Biodeterior. Biodegrad. 32. 75-85. Fielding. L.M.. Cook. P.E. and Grandison, A.S. (1994b) The effect of electron beam irradiation and modified pH on the survival and recovery of Escherictriu cdi. J. Appl. Bacteriol. 76. 412-416. Jay, J.M. (1992) Modern Food Microbiology, 4th edition. Van Nostrand. London, pp. 371. Quinn, D.J., Anderson, A.W. and Dyer, J.F. (1967) The inactivation of infection and intoxification micro-organisms by irradiation in seafood. In: Microbiological Problems in Food Preservation by Irradiation. Proc. Panel, Vienna, 27 June-l July 1966, organised by the Joint FAO! IAEA Division of Atomic Energy in Food and Agriculture. IAEA. Vienna, pp. I-13. Ray, B. (1992) The need for food biopreservation. In: B. Ray and M. Daeschel (editors), Food Biopreservatives of Microbial Origin. CRC Press, Boca Raton, FL. pp. I-23. Urbain. W.M. (1986) Food Irradiation. Academic Press, New York.