Journal of Applied Bacteriology 1990.69, 3W397
321 7110189
The influence of pH, temperature and organic acids on the initiation of growth of Yersinia enterocolitica T . F . B R O C K L E H U R& S TB A R B A R A M . L U N D AFRC Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich NR4 7UA U K Accepted 5 March 1990 B R O C K L E H U R S TT,. F . & LUND,B.M.1990. The influence of pH, temperature and organic acids on the initiation of growth of Yersinia enterocolitica. Journal of Applied Bacteriology 69, 390-397. The influence of incubation temperature, and of acetic, lactic and citric acids on the minimum pH for the initiation of growth of six strains of Yersinia enterocolitica was determined. The strains included two of serotype 0 : 9, two of serotype 0 : 3, and one each of serotypes 0 : 8 and 0 : 5.27. In a culture medium acidified with HCI to pH values between 4.0 and 6.0 at intervals of approximately 0.1 unit the minimum pH at which growth was detected after incubation at 20". lo", 7" and 4°C for 21 d was in the ranges 4,18436, 4.26-4.50, 4.36-4.83 and 4,42480, respectively. The minimum pH for growth was also determined in media that contained 17. 33 and 50 mmol/l acetic acid adjusted to pH values between 5.1 and 5.9 at intervals of approximately 0.2 unit, 24, 48 and 95 mmol/l citric acid adjusted to pH values between 4.1 and 4.9 at intervals of approximately 0.2 unit, and 22. 44. and 1 1 1 mmol/l lactic acid adjusted to pH values between 4.3 and 5.7 at intervals of approximately 0.4 or 0.5 unit. The effect of these concentrations of organic acids was, in most cases, to increase the minimum pH that allowed growth. The order of effectiveness of the organic acids in raising the minimum pH for growth was acetic > lactic > citric and the minimum inhibitory concentrations were greater at higher temperatures.
Yersinia enterocolitica has been implicated as a cause of food poisoning associated with the consumption of chocolate milk, tofu, pasteurized milk (Black er a/. 1978; Aulisio et al. 1983; Tacket er a / . 1984, 1985) and dissolved powdered milk and turkey chow mein (Shayegani et a/. 1983). It has been isolated from a wide range of foods (Swaminathan 1982). Although most strains were non-pathogenic 'environmental' strains, a small number of strains of those serotypes associated with human illness were isolated from raw pork products (Schiemann 1980). poultry products (De Boer et a/. 1982) and milk (Greenwood & Hooper i 1985). Environmental strains were also found in a wide range of foods in the Netherlands, although potentially virulent strains were isolated only from porcine tonsils (De Boer et al. 1986). It has been reported that pathogenic strains from clinical sources multiply in culture media at 2°C (Buckeridge et a / . 1980), 3°C (Stem et a / .
1980) and 4°C (Buckeridge et a/. 1980; Kendall & Gilbert 1980). on solid culture media at 4°C (Mossel e l a/. 1981), and in artificially contaminated raw or cooked foods of animal or vegetable origin stored at temperatures between &2"C and 10°C (Stern & Pierson 1979; Kendall & Gilbert 1980; Kovats et a/. 1984; Velin 1984). It is clearly possible, therefore, for pathogenic strains of Y . enrerocolitica to multiply in foods stored under refrigeration. In many chilled foods, however, the preservative effect of low temperature is used in combination with low pH and organic acids in order to increase the microbiological stability of the products. Although it has been reported that Y . enterocolirica survives in acidic environments, notably tartar sauce (cited by Stern & Pierson 1979) and artificially contaminated mayonnaise (Brackett 1986), there is little published information on the effects of temperature, pH, and organic acids on its growth.
39 1
Initiation of growth of Y.enterocolitica In a study of the effect of organic and mineral acids on the survival of three strains of Y. enrerocolirica, Brackett (1987) found that growth did not occur in trypticase soy broth (TSB) that contained either acetic, citric o r lactic acid (25 mmol/l) at pH 5.5, 4.0 and 5.0 respectively, or in TSB adjusted to pH 5.0 with HCL, during incubation at 25°C for 24 h. In other studies in culture media acidified by HCI, clinical strains multiplied at pH 4.4, but not at pH 4.2 during incubation at 22°C for 2 d (Kendall & Gilbert 1980), at pH 4.5, but not at pH 4.0 during incubation at 25°C for 2 d (Brackett 1986), and at pH 4.6, but not at pH 4.4 during incubation at 25°C for 12 h (Stern el al. 1980). Reports of studies at refrigeration temperatures are limited to findings that no growth occurred in a culture medium adjusted with HCI to pH 5.0 and incubated at 5°C (Brackett 1986). and that growth occurred in medium adjusted with HCI to pH 7.2 and incubated at 3"C, but not in this medium adjusted to pH 4.6 and incubated a t 3°C (Stern et af. 1980). The work reported here determined the minimum p H that allowed initiation of growth in culture media during incubation at 4", 7". 10" and 20°C for up to 21 d, and determined the effect of acetic, citric or lactic acids on this pH.
2 d and stored at 2°C. Inocula for studies of the effect of pH and organic acids on the initiation of growth were grown in TSB (Baltimore Biological Laboratory) (10 ml), and the determinations were made in TSB plus yeast extract (Difco), 0.3% (w/v) and glucose, 0.75% (w/v). This medium was prepared at 1 . 1 times or twice the final concentration. Media to determine the effect of organic acids on initiation of growth were made by the addition of appropriate volumes of stock solutions of acetic, citric or lactic acid. The pH of the media was measured at approximately 20°C. Medium without organic acids was adjusted with HCI. 1 mol/l, to pH values between 4.0 and 6.0, at intervals of approximately 0.1 unit. Media containing organic acids were adjusted with HCI 1 mol/l. or NaOH, 1 mol/l, to pH values between 5.1 and 5.9, at intervals of approximately 0.2 unit (for media containing acetic acid), to pH values between 4.1 and 4.9, at intervals of approximately 0.2 unit (for media containing citric acid), or to pH values between 4.3 and 5.7 at intervals of approximately 0.4 or 0.5 unit (for media containing lactic acid). The media were made up to the final concentration, and sterilized by filtration through a 0.22 y n membrane filter (Millipore UK) overlaid with a membrane filter with a pore size of 3 p n .
Materials and Methods D E T E R M I N A T I O N O F THE EFFECT OF
pH
BACTERIA
A N D O R G A N I C ACIDS ON THE INITIATION
Yersinia enrerocolirica strains YE 134, and BL 87/44 (serotype 0 : 3), strains YE 383 and BL 87/46 (serotype 0 : 9), strain YE 328 (serotype 0 : 5. 27), and strain BL 87/45 (serotype 0 : 8) were obtained from Mr E. Fox and Miss Delia Cope (Public Health Laboratory, Leicester Royal Infirmary, Leicester).
OF G R O W T H AT
CHEMICALS
Stock solutions were prepared that contained glacial acetic acid (BDH), 100 ml/l ( = 1.67 mol/l); analytical grade citric acid (BDH), 200 g/l ( ~ 9 5 2mmol/l); or L + lactic acid (Sigma), 1 0 0 g / l ( m l ~ l lmol/l). C U L T U R E MEDIA
Stock cultures were maintained on Heart Infusion Agar (Difco) slopes incubated at 25°C for
Zoo, l o " , 7" A N D 4°C
A technique similar to that described by Ferreira & Lund (1987) was used. T o prepare inocula, bacteria were grown for two successive cultures in TSB at 25"C, each for 24 h. The Aaso of the second culture was measured with a spectrophotometer (SP600, Pye Unicam) and the concentration of viable bacteria was determined from a calibration curve. A sample of the culture was diluted in peptone salt dilution fluid (Anon 1978) to give approximately 2.5 x lo6 viable bacteria/ml. Medium (240 PI) adjusted to each pH level, and with or without organic acids as required, was dispensed into each of eight replicate wells in each microplate. The eight replicate wells at each end of the plate contained sterile glass distilled water (GDW; 250 PI). The wells that contained G D W were not inoculated with bacteria,
392
T . F . Brocklehurst and Barbara M . Lund
and of the remaining eight replicate wells of each medium the central four received 10 pI of the inoculum. This resulted in approximately 2.5 x lo4 bacteria/well of the microplate, equivalent to lo5 viable bacteria/ml. Inoculated plates were incubated as described by Ferreira & Lund (1987). During incubation, growth was determined periodically either by visual examination, or by measurement of the absorption of the medium in each well using a microplate reader (MR 700, Dynatech Laboratories Ltd, Sussex, UK) with the 630 nm filter. Measurements were made with the lid of the microplate in position, and precautions were taken to minimize any change in temperature of the microplate. Where condensation on the lid obscured the light path of the plate reader, the lid was removed aseptically, and a replacement sterile lid (Sterilin) substituted. All bacteria tested sedimented during growth. After incubation of the plates for 21 d the sedimented cells in the wells were resuspended by agitation of the culture using a fine glass rod before determination of growth in the plate reader. Growth of bacteria in the microplates was recorded as positive if the growth was visible, or if the mean A,,, of the four inoculated wells was at least 0.025 unit higher than that of the four corresponding uninoculated wells. This was equivalent to an increase of approximately 800-fold in the number of viable bacteria/ml. All experiments were performed in duplicate. MEASUREMENT OF
pH
The pH of the media was measured with a Philips digital pH meter (PW 9410) and combined glass and reference electrode (Pye Unicam, Type CE2). Measurements were made at the time of inoculation of microplates (in medium remaining after the wells of the microplate had beem loaded), and at the end of experiments when medium was decanted from uninoculated wells into narrow glass tubes.
MEASUREMENT OF T H E CONCENTRATION OF ACETIC A N D LACTIC A C I D S
Samples of medium that contained 83.3 mmol/l acetic acid or 55.5 mmol/l lactic acid were analysed before and after sterilization by fil-
tration, and after incubation in microplates at 20°C for 7 and 21 d. In these the medium was removed by pipette from each of the central four wells of uninoculated plates, and pooled. The concentration of acetic and lactic acids remaining in the media was determined by HPLC. The apparatus consisted of a model 7125 Rheodyne injector (Anachem) fitted with a 20 pI injection loop, a SA 6410 pump (Severn Analytical) and a stainless steel column (300 mm x 7.8 mm i.d.) packed with Aminex HPX-87H (Biorad) protected by a Microguard cation H + guard column (Biorad). The eluent was H, SO,, 4 mmol/l, and the flow rate was I ml/min. Acetic and lactic acids were detected by absorbance at 210 nm using a model LC75 variable wavelength ultraviolet detector (Perkin Elmer). The retention times of acetic and lactic acid were approximately 9.4 and 7.9 min, respectively. The mean recovery of acetic and lactic acids was 95.8%, S.D. 3.3%, n = 4, and 99.9%. S.D. 1.8%. n = 4, respectively.
Results After incubation for 21 d the pH in uninoculated wells adjacent to wells that did not show growth was usually within f0.02 pH unit of the initial pH. Measurement of the concentration of organic acids in the media showed that lactic acid was not lost during sterilization by filtration or during incubation of microplates at 20°C for 21 d. Acetic acid was not lost during sterilization by filtration, but the concentration of acetic acid in the central four wells of uninoculated plates loaded with medium that contained 83.3 mmol acetic acid/l and adjusted to pH 4.0, 4.5, 5.0 and 5.5 decreased by 25, 7, 2 and 2%, respectively, after incubation at 20°C for 21 d. The effect of temperature and time of incubation on the minimum pH, adjusted with HCl, that allowed growth, and the influence of acetic, citric or lactic acids on the minimum pH for growth, of representative strains is shown in Tables I, 2, 3, and 4. The minimum pH at which growth of all strains tested was detected after incubation at 20°, lo", 7" and 4°C for 21 d was in the ranges 4.18436, 4.26-4.50, 4 . 3 6 4.83 and 4.504430, respectively. The effect of organic acids at the concentrations tested was,
Initiation of growth of Y.enterocolitica
393
TaMe 1, The effect of incubation time, temperature and the presence of organic acids on the minimum pH for growth of Yersiniu enterocoliticu BL 87/44 (serotype 0 : 3)
Organic acid
Growth after incubation for (d): 8
Temperature ("C)
Type
20
Acetic Citric Lactic 10
Acetic Citric Lactic 7 4
Acetic Citric Lactic
Concn. (mmol/l) 0 17 33 505 24 48 9511 22 44 iiin 0 17 33 50
24 48 95 22 44 Ill 0 0 17 33 50 24 48 95
22 44 Ill
14
21
+
-
+
-
+
-
4.36 5.55 5.72
4.28 5.40 5.5 I 5.71 4.17 4.36 4.36 4.39 4.8 I 5.22 4.50 5.75 5.72 5.71 4.58 4.76 4.75 4.82 4.8 1 5.22 4.90 5.28 5.75 5.72 5.71 4.80 4.76 4.75 5.21 5.22 5.22
4.36
4.28 z
4.36 5.20 5.30
4.28
z z
%
4.38 4.56 4.55 4.82 5.22 5.58 4.65
%
3
%
4.80 5.21 5.22 5.58 5.03 5.42
3 % %
3 3 1
5.62 5.62 5.58
z
z
z
4.38 4.36 4.55 4.82 5.22 5.58 4.65
4.17 4.17 4.36 4.39 4.8 I 5.22 4.50
z z
*
z z z
4.58 4.76 4.75 4.82 5.22 5.58 4.90 5.16
4.38 4.56 4.55 4.39 4.81 5.22 4.83 5.03
z z
3
% %
5.21 5.22 5.58
4.80 4.76 4.75 4.82 4.8 1 5.22
3
4.38 4.36 4.55 4.82 5.22 5.58 4.50 5.75
3 3 4.58 4.76 4.75 4.82 5.22 5.58 4.83 4.80
1 3 3 4.80 4.76
% 5.21 5.22 5.58
t 5.08 5.71 4.17 4.17 4.36 4.39 4.8 I 5.22 4.36 5.55
5.72 5.71 4.38 4.56 4.55 4.39 4.8 I 5.22 4.73 4.65 5.75 5.72 5.71 4.58 4.56 4.75 4.82 4.8 I 5.22
Not tested.
t The pH at which growth occurred after incubation for 21 d was the lowest pH tested. 3 A pH higher than that at which no growth occurred was not tested. 4 = acetic acid, 0.3% (w/v). 11 = citric acid, 2.0% (w/v). 7 = lactic acid, 1.0% (w/v). in most cases, to increase the minimum pH that allowed growth. The minimum concentration of organic acids that inhibited growth of Y.enrerocolitica could not be determined reliably where no growth occurred at the highest pH tested. Where growth occurred, however, the calculated minimum concentration of undissociated acetic and lactic acids that inhibited growth after incubation for 21 d was (mmol/l): acetic acid, 2.37 at 10"C, and in the range (depending on strain) 4.9810.68 at 20°C; lactic acid, in the ranges (depending on strain) 4.35485 at 4"C, 4 . 2 6 10.97 at 10°C and 4.17-10.74 at 20°C.
Mscdoa In culture medium acidified with HCI the lowest pH at which Y.enterocolitica multiplied at 20°C in 21 d was pH 4.18, and no growth was detected at pH 3.95 (Table 3). Similar limiting pH values during incubation at 22°C and 25°C have been reported by other workers (Kendall & Gilbert 1980; Brackett 1986). Our results also show the ability of this bacterium to grow in acidic conditions at low temperature; at lo", 7" and 4°C multiplication occurred at pH 4.26, pH 4.36 and pH 4.50 respectively in 21 d. Measurement of the concentration of acetic acid in uninoculated media showed that losses
T . F . Brocklehurst and Barbara M . Lund
394
Table 2. The effect of incubation time, temperature and the presence of organic acids on the minimum pH for growth of Yersinia enterocolitico strain BL 87/46 (serotype 0 : 9) Growth after incubation for (d):
Organic acid 8
Temperature ("C)
TYPe
20
Acetic Citric Lactic
Concn. (mmol/l) 0 17 33 5ori 24 48 9511 22
44 illy 10
Acetic Citric Lactic 7 4
Acetic
0 17 33 50 24 48 95 22
44 Ill 0 0 17 33 50
Citric Lactic
24 48 95 22
44 Ill
14
21
+
-
+
-
+
-
4.28
4.20 5.40
4.20
4.09
5.72
5.51
4.20 5.20 5.72
4.09
5.55
%
5.7 I 4.17 4.36 4.36 4.39 4.81 5.22 4.36 5.75
4.38 4.56 4.55 4.82 5.22 5.58 4.50
% % %
4.58 4.76 4.75 4.82 5.22 5.58
%
5.28
% %
1 % % %
5.21 5.62 5.58
5.72 5.71 4.38 4.56 4.55 4.39 4.81 5.22 5.95 5.16 5.75 5.72 5.7 I 4.80 4.76 4.75 4.82 5.22 5.22
* 4.17 4.36 4.55 4.82 5.22 5.58 4.36
4.38 4.56 4.75 4.82 5.22 5.58
1
4.80
* 4.80 4.76
%
4.82 5.22 5.58
t 4.17 4.36 4.39 4.8 I 5.22 4.28
4.17 4.36 4.55 4.39 4.8 I 5.22 5.95 4.62
*
* 4.58 4.56 4.75 4.39 4.81 5.22
% 4.17 4.36 4.55 4.82 4.8 I 5.22 4.36 5.75
% %
4.38 4.56 4.55
4.82 4.8 I 5.22 4.73 4.62
% % %
4.58 4.76 4.75 4.82 5.22 5.58
t 5.5 I 5.71
t 4.17 4.36 4.39 4.4 I 4.83 4.28 5.55
5.72 5.71 4.17 4.36 4.36 4.39 4.4 I 4.83 4.50 4.50 5.75 5.72 5.71 4.38 4.56 4.55 4.39 4.8 I 5.22
Not tested.
t The pH at which growth occurred after incubation for 21 d was the lowest pH tested. 3 A pH higher than that at which no growth occurred was not tested.
= acetic acid, 0.3% (w/v) 11 =citric acid, 2.0% (w/v). = lactic acid. 1.0% (w/v).
(i
occurred during incubation of microplates. The loss increased with time and was greater in medium adjusted to pH 4.0 than in media adjusted to pH 5.0 or 5.5. It was probably due to loss of the volatile undissociated form of acetic acid. In the work reported here the lowest pH at which growth occurred in the presence of acetic acid was pH 5.20. At this and at higher pH values the loss of acetic acid from microplates incubated at 20°C for 21 d was < 2% of the initial concentration. Lactic acid was not lost throughout incubation of uninoculated microplates at 20°C for 21 d. Citric acid is also a non-volatile acid and
it was assumed that no loss of this acid occurred. The effect of acetic, citric or lactic acid in the medium was, in most cases, to increase the minimum pH for the initiation of growth. In the case of lactic acid this increase was between 0.2 and 0.7 pH unit in the presence of 22 mmol/l lactic acid, between 0.4 and 1.1 pH unit in the presence of 44 mmol/l lactic acid, and between 0.8 and 1.4 pH unit in the presence of I 1 1 mmol/l lactic acid, after incubation at 20°C for 21 d. The effect of acetic acid was more marked, and the increase in the minimum pH for initi-
Initiation of growth of Y.enterocolitica
395
Table 3. The effect of incubation time, temperature and the presence of organic acids on the minimum pH for growth of Yersiniu enterocoliticu strain YE 328 (serotype 0 : 5, 27) Growth after incubation for (d):
Organic acid 8
Temperature ("C)
Type
20
Acetic Citric Lactic
Concn. (mmol/l) 0 17 33 5% 24 48 95 II 22 44
lllq 10
Acetic Citric Lactic 7 4
Acetic Citric Lactic
0 17 33 50 24 48 95 22 44
Ill 0 0 17 33 50 24 48 95 22 44 Ill
14
21
+
-
+
-
+
-
4.26 5.40 5.72
4.18 5.20 5.51 5.71 4.24 4.28 4.48 4.45 4.84 4.83 4.36 5.75 5.72 5.71 4.47 4.48 4.69 4.87 4.84 5.24 4.62 5.18 5.75 5.72 5.71 4.87 4.85 4.88 4.87 5.29 5.24
4.18 5.40 5.72
3.95 5.20 5.51 5.71 4.24 4.28 4.48
4.18 5.20 5.30
3.95
%
4.47 4.48 4.69 4.87 5.29 5.24 4.48
% % t
4.66 4.68 4.88 5.25 5.29 5.66 4.8 I 5.27
% % % % % %
5.25 5.66 5.66
1
4.47 4.48 4.69
* * 4.36 5.75
% %
4.66 4.68 4.69
4.26 5.55 5.72 5.71 4.47 4.48 4.48
* *
4.62 4.92
% % %
4.87 4.85
%
4.48 4.8 1 5.75 5.72 5.71 4.66 4.68 4.88
%
4.47 4.48 4.69 4.87 5.29 5.24 4.26 5.75
% % 4.47 4.68 4.69 4.87 5.29 5.66 4.36 4.62
1
% %
4.87 4.85
%
4.87 5.29 5.66
t 5.08 5.71 4.24 4.28 4.48 4.45 4.84 4.83 4.18 5.55 5.72 5.71 4.24 4.48 4.48 4.45 4.84 5.24 4.26 4.48 5.75 5.72 5.71 4.66 4.68 4.88 4.45 4.84 5.24
* Not tested.
t The pH at which growth occurred after incubation for 21 d was the lowest pH tested. 1A pH higher than that at which no growth occurred was not tested. 8 = acetic acid, 0.3% (w/v) 11 =citric acid, 2.0% (w/v).
T
= lactic acid, 1.0% (w/v).
ation of growth was greater than in the presence of similar total (i.e. undissociated dissociated) concentrations of lactic acid. This increase was between 0.8 and 1.5 pH unit in the presence of 17 mmol/l acetic acid, and between 0.9 and 1.5 pH unit in the presence of 33 mmol/l acetic acid, after incubation at 20°C for 21 d. The effect of citric acid was less marked than that of either acetic or lactic acid. In most cases the minimum pII for the initiation of growth at 20", 10" and 4°C in the presence of 24 mmol/l citric acid was similar to the limiting pH in the absence of citric acid. At the highest concentration tested (95 mmol/l citric acid) an increase in
+
the limiting pH of up to 0.5 unit occurred at 20°C. Growth did not occur in the presence of 50 mmol/l acetic acid at pH 5.71 at any temperature tested, in the presence of 33 mmol/l acetic acid at pH 5.72 and lo" or 4"C, at pH 5.08 or 5.51 and 20°C. and in the presence of 17 mmol/l acetic acid at pH 5.75 and 4"C, and at pH 5.55 and 10°C. Growth was inhibited by lactic acid at a concentration of 22 mmol/l at pH 4.39-4.82, at 4°C. and between pH 4.39 and 4.45 at 10" and 20°C; at a concentration of 44 mmol/l at pH 4.814.84 at 4"C, and at pH 4 . 4 1 4 8 4 at 10"
T . F. Brocklehurst and Barbara M . Lund
396
Table 4. The effect of incubation time. temperature and the presence of organic acids on the minimum pH for growth of Yersinio errrerocolirica strain BL 87/45 (serotype 0 : 8) Organic acid
Growth after incubation for (d): 8
Temperature ("C)
14
21
Type
Concn. (mmol/l)
+
-
+
-
+
-
0 17 33
4.20 5.40 5.72
4.09 5.20 5.51 5.71 4.17 4.36 4.36 4.39 4.81 5.22 4.28 5.75 5.72 5.71 4.17 4.36 4.36 4.39 4.81 5.22 4.50
4.20
4.09
4.20 5.20
4.09
Acetic
5.30
5.08 5.7 1
20
%
5w Citric Lactic
24 48 95 II 22 44
1117 0
10
Acetic Citric Lactic 7 4
Acetic Citric Lactic
17 33 50 24 48 95 22 44 111 0 0 17 33 50 24 48 95 22 44 111
4.38 4.56 4.55 4.82 5.22 5.58 4.36
% % 1
4.38 4.56 4.55 4.82 5.22 5.58 4.65 5.16
% % 1 1 % %
5.2 1 5.22 5.58
5.03 5.75 5.72 5.71 4.80 4.76 4.75 4.82 4.81 5.22
* *
4.17 4.36 4.55 4.82 4.81 5.58 4.28
*
4.38 4.36 4.55 4.82 4.81 5.58 4.50 4.65
4.58 4.56
1
4.82 5.22 5.58
t 4.17 4.36 4.39 4.41 5.22 4.20
4.17 4.17 4.36 4.39 4.41 5.22 4.36 4.50
4.38 4.36 4.75 4.39 4.81 5.22
%
4.17 4.17 4.55 4.82 4.81 5.58 4.28 5.75
% %
4.38 4.36 4.55 4.82 4.81 5.58 4.36 4.50
1 % %
4.38 4.56 4.55 4.82 5.22 5.58
t
t t 4.36 4.39 4.4 1 5.22 4.20 5.55 5.72 5.71 4.17 4.17 4.36 4.39 4.41 5.22 4.28 4.36 5.75 5.72 5.71 4.17 4.36 4.36 4.39 4.8 1 5.22
Not tested.
t The pH at which growth occurred after incubation for 21 d was the lowest pH tested. 1A pH higher than that at which no growth occurred was not tested.
acetic acid. 0.3% (w/v) . . _ icitric acid..2.0% (w/v). 7 i lactic acid, 1.0% (w/v). 6
i
and 20°C; and at a concentration of 11 1 mmol/l at pH 5.22-5.24 at 4"C, at pH 4.83-5.24 at 10°C and at pH 4433-5.22 at 20°C. The antimicrobial activity of weak acids is due mainly to the concentration of undissociated acid (Baird-Parker 1980; Eklund 1985). The concentration of undissociated molecules varies according to the total concentration of acid, the pH and the dissociation constant (pK), which varies with temperature (Robinson & Stokes 1959). In those cases where no growth occurred at the highest pH tested a reliable determination of the inhibitory concentration of undissociated
acid could not be made. If these data are excluded, the calculated inhibitory concentrations of undissociated acetic acid (assuming pK,, = 4.76, pK,, = 4.76 and pK, = 4.77) and lactic acid (assuming pK,, = 3.86, pK,, = 3.87 and pK, = 3.88) were within the ranges found to be lethal to Y. enterocolitica by Brackett (1987). The calculated inhibitory concentrations of undissociated lactic acid were also in agreement with the work of Grau (1981) who found that an 'environmental' strain multiplied in the presence of 3 mmol/l ( = a total concentration of 150 mmol/l at pH 5.55) in culture medium incubated aerobically at 5" or 25°C although this
Initiation of growth of Y.enterocolitica concentration was inhibitory t o cultures incubated anaerobically. W e are grateful t o M r A. Hobson-Frohock a n d Miss Angela Bailey for their collaboration in setting up assays for acetic a n d lactic acids, a n d t o Miss A.J. Thompson for technical assistance. This work was funded by the Ministry of Agriculture. Fisheries a n d Food.
References ANON.1978 Microorganisms in Foodr. 1 . Their Significance and Methodr of Enumeration, 2nd edn. International Commission for the Microbiological Specifications for Foods. Toronto: University of Toronto Press. AULISIO,C.C.G., STANFIELD, J.T., WEAGANT, S.D. & HILL, W.E. 1983 Yersiniosis associated with tofu consumption : serological, biochemical and pathogenicity studies of Yersinia enterocolitica isolates. Journal of Food Protection 46.226-230. BAIRD-PARKER, A.C. 1980 Organic acids. In Microbial Ecology of Fooh, Vol. I . Factors Affecting Lve and Death of Microorganisms. pp. 126-135. The International Commission on Microbiological Specifications for Foods. New York: Academic Press. BLACK,R.E., JACKSON,R.J., TSAI,T.,MEDVTSKY,M., SHAYEGANI,M.,FEELEY, J.C., MACLEOD.K.I.E. & WAKELEE,A.M. 1978 Epidemic Yersinia enterocolitica infection due to contaminated chocolate milk. The New England Journal of Medicine 290. 76-79.
BRACKETT, R.E. 1986 Growth and survival of Yersinia enterocolitica at acidic pH. International Journal of Food Microbiology 3,243-251. BRACKETT,R.E. 1987 Effects of various acids on growth and survival of Yersinia enterocolitica. Journal ofFood Protection 50,598401.
BUCKERIDGE, S.A., SEAMAN, M.A. & WOODBINE, M. 1980 Effects of temperature and carbohydrate on the growth and survival of Yersinia enterocolitica. In Microbial Growth and Survival in Extremes of Environment. Society for Applied Bacteriology Technical Series No. 17. ed. Gould, G.W. & Corry, J.E.L. pp. 205-214. London: Academic Press. J. M 1982 , DE Bow, E., HARMG, B.J. & O ~ ~ T E R O Occurrence of Yersinia enterocolitica in poultry products. Journal of Food Protection 45, 322-325. DE Bow, E., SELDAM,W.M. & OOSTEROM, J. 1986 Characterisation of Yersinia enterocolitica and related species isolated from foods and porcine tonsils in the Netherlands. International Journal of Food Microbiology 3, 217-224. EKLUND.T. 1985 Inhibition of microbial growth at different pH levels by benzoic and propionic acid and esters of phydroxybenzoic acid. International Journal of Food Microbiology 2, 159-168. FERREIRA,M.A.S.S. & L m B.M. 1987 The influence
397
of pH and temperature on initiation of growth of Salmonella spp. Letters in Applied Microbiology 5, 67-70. GRAU,F.H. 1981 Role of pH lactate and anaerobiosis in controlling the growth of some fermentative Gram-negative bacteria on beef. Applied and Environmental Microbiology 42, 1043-1050. GREENWOOD, M.H. & HOOPER,W.L. 1985 Yersinia spp. in foods and related environments. Food Microbiology 2, 263-269. KENDALL,M. & GILBERT,R.J. 1980 Survival and growth of Yersinia enterocolitica in broth media and in food. In Microbial Growth and Survival in extremes of Environment. Society for Applied Bacteriology Technical Series No. 17. ed. GOULD,G.W. CORRY,J.E.L. pp. 2 15-266. London : Academic
Press. N. 1984 KOVATS, S.K.. DOYLE,M.P. & TANAKA, Evaluation of the microbiological safety of tofu. Journal ofFood Protection 41,618-622.
MOSSEL,D.A.A., JANSMA,M. & DE WAART,J. 1981 Growth potential of 114 strains of epidemiologically most common salmonellae and arizonae between 3 and 17°C. In Psychrotrophic Microorganisms in Spoilage and Pathogenicity. ed. ROBERTS,T.A., Hones, G., CHRISTIAN,J.H.B. & SKORGMRD,N. pp. 29-37. London: Academic Press. ROBINSON, R.A. & STOKES, R.H. 1959 Electrolyte Solutions. pp. 357-358. London: Butterworths. SCHIEMANN, D.A. 1980 Isolation of toxigenic Yersinia enrerocolitica from retail pork products. Journal of Food Protection 43, 360-365. SHAYEGANI, M.,MORSE, D., DEFORGE, I., ROOT, T., PARSONS,L.M. & MAUPIN,P.S. 1983 Microbiology
of a major foodbome outbreak of gastroenteritis caused by Yersinia enterocolitica serogroup 0 : 8. Journal of Clinical Microbiology 17, 35-40. STERN,N.J. & PIERSON, M.D. 1979 Yersinia enterocolitica: a review of the psychrotrophic water and foodbome pathogen. Journal of Food Science 44. 1736-1 742.
STWIN,N.J., PIERSON, M.D. & KOTULA,A.W. 1980 Effect of pH and sodium chloride on Yersinia entercolitica growth at room and refrigeration temperatures. Journal of Food Science 45,6447. SWAMINATHAN, B., HARMON,M.C. & MEHLMAN,I.J. 1982 A review. Yersinia enterocolitica. Journal of Applied Bacteriology 52, 151-183.
TACKET,C.O., BALLARD,J. & HARRIS,N. 1985 An outbreak of Yersinia enterocolitica infections caused by contaminated tofu (soybean curd). American Journal of Epidemiology 121,705-71 1. TACKET,C.O., NARAIN,J.P., SATTIN, R., LOFGREN, J.P., KONIGSBERG, C., RENDTORFF,R.C., RAUSA, A., DAVIS,B.R. & COHEN,M.L. 1984 A multistate outbreak of infections caused by Yersinia enterocolitica transmitted by pasteurized milk. Journal of the American Medical Association 251,483486.
VELIN,D. 1984 Enterotoxin production by Yersinia enterocolitica in food samples. Acta microbiologica Academiae scientarum hungaricae 31,4348.
321 7110189
The influence of pH, temperature and organic acids on the initiation of growth of Yersinia enterocolitica T . F . B R O C K L E H U R& S TB A R B A R A M . L U N D AFRC Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich NR4 7UA U K Accepted 5 March 1990 B R O C K L E H U R S TT,. F . & LUND,B.M.1990. The influence of pH, temperature and organic acids on the initiation of growth of Yersinia enterocolitica. Journal of Applied Bacteriology 69, 390-397. The influence of incubation temperature, and of acetic, lactic and citric acids on the minimum pH for the initiation of growth of six strains of Yersinia enterocolitica was determined. The strains included two of serotype 0 : 9, two of serotype 0 : 3, and one each of serotypes 0 : 8 and 0 : 5.27. In a culture medium acidified with HCI to pH values between 4.0 and 6.0 at intervals of approximately 0.1 unit the minimum pH at which growth was detected after incubation at 20". lo", 7" and 4°C for 21 d was in the ranges 4,18436, 4.26-4.50, 4.36-4.83 and 4,42480, respectively. The minimum pH for growth was also determined in media that contained 17. 33 and 50 mmol/l acetic acid adjusted to pH values between 5.1 and 5.9 at intervals of approximately 0.2 unit, 24, 48 and 95 mmol/l citric acid adjusted to pH values between 4.1 and 4.9 at intervals of approximately 0.2 unit, and 22. 44. and 1 1 1 mmol/l lactic acid adjusted to pH values between 4.3 and 5.7 at intervals of approximately 0.4 or 0.5 unit. The effect of these concentrations of organic acids was, in most cases, to increase the minimum pH that allowed growth. The order of effectiveness of the organic acids in raising the minimum pH for growth was acetic > lactic > citric and the minimum inhibitory concentrations were greater at higher temperatures.
Yersinia enterocolitica has been implicated as a cause of food poisoning associated with the consumption of chocolate milk, tofu, pasteurized milk (Black er a/. 1978; Aulisio et al. 1983; Tacket er a / . 1984, 1985) and dissolved powdered milk and turkey chow mein (Shayegani et a/. 1983). It has been isolated from a wide range of foods (Swaminathan 1982). Although most strains were non-pathogenic 'environmental' strains, a small number of strains of those serotypes associated with human illness were isolated from raw pork products (Schiemann 1980). poultry products (De Boer et a/. 1982) and milk (Greenwood & Hooper i 1985). Environmental strains were also found in a wide range of foods in the Netherlands, although potentially virulent strains were isolated only from porcine tonsils (De Boer et al. 1986). It has been reported that pathogenic strains from clinical sources multiply in culture media at 2°C (Buckeridge et a / . 1980), 3°C (Stem et a / .
1980) and 4°C (Buckeridge et a/. 1980; Kendall & Gilbert 1980). on solid culture media at 4°C (Mossel e l a/. 1981), and in artificially contaminated raw or cooked foods of animal or vegetable origin stored at temperatures between &2"C and 10°C (Stern & Pierson 1979; Kendall & Gilbert 1980; Kovats et a/. 1984; Velin 1984). It is clearly possible, therefore, for pathogenic strains of Y . enrerocolitica to multiply in foods stored under refrigeration. In many chilled foods, however, the preservative effect of low temperature is used in combination with low pH and organic acids in order to increase the microbiological stability of the products. Although it has been reported that Y . enterocolirica survives in acidic environments, notably tartar sauce (cited by Stern & Pierson 1979) and artificially contaminated mayonnaise (Brackett 1986), there is little published information on the effects of temperature, pH, and organic acids on its growth.
39 1
Initiation of growth of Y.enterocolitica In a study of the effect of organic and mineral acids on the survival of three strains of Y. enrerocolirica, Brackett (1987) found that growth did not occur in trypticase soy broth (TSB) that contained either acetic, citric o r lactic acid (25 mmol/l) at pH 5.5, 4.0 and 5.0 respectively, or in TSB adjusted to pH 5.0 with HCL, during incubation at 25°C for 24 h. In other studies in culture media acidified by HCI, clinical strains multiplied at pH 4.4, but not at pH 4.2 during incubation at 22°C for 2 d (Kendall & Gilbert 1980), at pH 4.5, but not at pH 4.0 during incubation at 25°C for 2 d (Brackett 1986), and at pH 4.6, but not at pH 4.4 during incubation at 25°C for 12 h (Stern el al. 1980). Reports of studies at refrigeration temperatures are limited to findings that no growth occurred in a culture medium adjusted with HCI to pH 5.0 and incubated at 5°C (Brackett 1986). and that growth occurred in medium adjusted with HCI to pH 7.2 and incubated at 3"C, but not in this medium adjusted to pH 4.6 and incubated a t 3°C (Stern et af. 1980). The work reported here determined the minimum p H that allowed initiation of growth in culture media during incubation at 4", 7". 10" and 20°C for up to 21 d, and determined the effect of acetic, citric or lactic acids on this pH.
2 d and stored at 2°C. Inocula for studies of the effect of pH and organic acids on the initiation of growth were grown in TSB (Baltimore Biological Laboratory) (10 ml), and the determinations were made in TSB plus yeast extract (Difco), 0.3% (w/v) and glucose, 0.75% (w/v). This medium was prepared at 1 . 1 times or twice the final concentration. Media to determine the effect of organic acids on initiation of growth were made by the addition of appropriate volumes of stock solutions of acetic, citric or lactic acid. The pH of the media was measured at approximately 20°C. Medium without organic acids was adjusted with HCI. 1 mol/l, to pH values between 4.0 and 6.0, at intervals of approximately 0.1 unit. Media containing organic acids were adjusted with HCI 1 mol/l. or NaOH, 1 mol/l, to pH values between 5.1 and 5.9, at intervals of approximately 0.2 unit (for media containing acetic acid), to pH values between 4.1 and 4.9, at intervals of approximately 0.2 unit (for media containing citric acid), or to pH values between 4.3 and 5.7 at intervals of approximately 0.4 or 0.5 unit (for media containing lactic acid). The media were made up to the final concentration, and sterilized by filtration through a 0.22 y n membrane filter (Millipore UK) overlaid with a membrane filter with a pore size of 3 p n .
Materials and Methods D E T E R M I N A T I O N O F THE EFFECT OF
pH
BACTERIA
A N D O R G A N I C ACIDS ON THE INITIATION
Yersinia enrerocolirica strains YE 134, and BL 87/44 (serotype 0 : 3), strains YE 383 and BL 87/46 (serotype 0 : 9), strain YE 328 (serotype 0 : 5. 27), and strain BL 87/45 (serotype 0 : 8) were obtained from Mr E. Fox and Miss Delia Cope (Public Health Laboratory, Leicester Royal Infirmary, Leicester).
OF G R O W T H AT
CHEMICALS
Stock solutions were prepared that contained glacial acetic acid (BDH), 100 ml/l ( = 1.67 mol/l); analytical grade citric acid (BDH), 200 g/l ( ~ 9 5 2mmol/l); or L + lactic acid (Sigma), 1 0 0 g / l ( m l ~ l lmol/l). C U L T U R E MEDIA
Stock cultures were maintained on Heart Infusion Agar (Difco) slopes incubated at 25°C for
Zoo, l o " , 7" A N D 4°C
A technique similar to that described by Ferreira & Lund (1987) was used. T o prepare inocula, bacteria were grown for two successive cultures in TSB at 25"C, each for 24 h. The Aaso of the second culture was measured with a spectrophotometer (SP600, Pye Unicam) and the concentration of viable bacteria was determined from a calibration curve. A sample of the culture was diluted in peptone salt dilution fluid (Anon 1978) to give approximately 2.5 x lo6 viable bacteria/ml. Medium (240 PI) adjusted to each pH level, and with or without organic acids as required, was dispensed into each of eight replicate wells in each microplate. The eight replicate wells at each end of the plate contained sterile glass distilled water (GDW; 250 PI). The wells that contained G D W were not inoculated with bacteria,
392
T . F . Brocklehurst and Barbara M . Lund
and of the remaining eight replicate wells of each medium the central four received 10 pI of the inoculum. This resulted in approximately 2.5 x lo4 bacteria/well of the microplate, equivalent to lo5 viable bacteria/ml. Inoculated plates were incubated as described by Ferreira & Lund (1987). During incubation, growth was determined periodically either by visual examination, or by measurement of the absorption of the medium in each well using a microplate reader (MR 700, Dynatech Laboratories Ltd, Sussex, UK) with the 630 nm filter. Measurements were made with the lid of the microplate in position, and precautions were taken to minimize any change in temperature of the microplate. Where condensation on the lid obscured the light path of the plate reader, the lid was removed aseptically, and a replacement sterile lid (Sterilin) substituted. All bacteria tested sedimented during growth. After incubation of the plates for 21 d the sedimented cells in the wells were resuspended by agitation of the culture using a fine glass rod before determination of growth in the plate reader. Growth of bacteria in the microplates was recorded as positive if the growth was visible, or if the mean A,,, of the four inoculated wells was at least 0.025 unit higher than that of the four corresponding uninoculated wells. This was equivalent to an increase of approximately 800-fold in the number of viable bacteria/ml. All experiments were performed in duplicate. MEASUREMENT OF
pH
The pH of the media was measured with a Philips digital pH meter (PW 9410) and combined glass and reference electrode (Pye Unicam, Type CE2). Measurements were made at the time of inoculation of microplates (in medium remaining after the wells of the microplate had beem loaded), and at the end of experiments when medium was decanted from uninoculated wells into narrow glass tubes.
MEASUREMENT OF T H E CONCENTRATION OF ACETIC A N D LACTIC A C I D S
Samples of medium that contained 83.3 mmol/l acetic acid or 55.5 mmol/l lactic acid were analysed before and after sterilization by fil-
tration, and after incubation in microplates at 20°C for 7 and 21 d. In these the medium was removed by pipette from each of the central four wells of uninoculated plates, and pooled. The concentration of acetic and lactic acids remaining in the media was determined by HPLC. The apparatus consisted of a model 7125 Rheodyne injector (Anachem) fitted with a 20 pI injection loop, a SA 6410 pump (Severn Analytical) and a stainless steel column (300 mm x 7.8 mm i.d.) packed with Aminex HPX-87H (Biorad) protected by a Microguard cation H + guard column (Biorad). The eluent was H, SO,, 4 mmol/l, and the flow rate was I ml/min. Acetic and lactic acids were detected by absorbance at 210 nm using a model LC75 variable wavelength ultraviolet detector (Perkin Elmer). The retention times of acetic and lactic acid were approximately 9.4 and 7.9 min, respectively. The mean recovery of acetic and lactic acids was 95.8%, S.D. 3.3%, n = 4, and 99.9%. S.D. 1.8%. n = 4, respectively.
Results After incubation for 21 d the pH in uninoculated wells adjacent to wells that did not show growth was usually within f0.02 pH unit of the initial pH. Measurement of the concentration of organic acids in the media showed that lactic acid was not lost during sterilization by filtration or during incubation of microplates at 20°C for 21 d. Acetic acid was not lost during sterilization by filtration, but the concentration of acetic acid in the central four wells of uninoculated plates loaded with medium that contained 83.3 mmol acetic acid/l and adjusted to pH 4.0, 4.5, 5.0 and 5.5 decreased by 25, 7, 2 and 2%, respectively, after incubation at 20°C for 21 d. The effect of temperature and time of incubation on the minimum pH, adjusted with HCl, that allowed growth, and the influence of acetic, citric or lactic acids on the minimum pH for growth, of representative strains is shown in Tables I, 2, 3, and 4. The minimum pH at which growth of all strains tested was detected after incubation at 20°, lo", 7" and 4°C for 21 d was in the ranges 4.18436, 4.26-4.50, 4 . 3 6 4.83 and 4.504430, respectively. The effect of organic acids at the concentrations tested was,
Initiation of growth of Y.enterocolitica
393
TaMe 1, The effect of incubation time, temperature and the presence of organic acids on the minimum pH for growth of Yersiniu enterocoliticu BL 87/44 (serotype 0 : 3)
Organic acid
Growth after incubation for (d): 8
Temperature ("C)
Type
20
Acetic Citric Lactic 10
Acetic Citric Lactic 7 4
Acetic Citric Lactic
Concn. (mmol/l) 0 17 33 505 24 48 9511 22 44 iiin 0 17 33 50
24 48 95 22 44 Ill 0 0 17 33 50 24 48 95
22 44 Ill
14
21
+
-
+
-
+
-
4.36 5.55 5.72
4.28 5.40 5.5 I 5.71 4.17 4.36 4.36 4.39 4.8 I 5.22 4.50 5.75 5.72 5.71 4.58 4.76 4.75 4.82 4.8 1 5.22 4.90 5.28 5.75 5.72 5.71 4.80 4.76 4.75 5.21 5.22 5.22
4.36
4.28 z
4.36 5.20 5.30
4.28
z z
%
4.38 4.56 4.55 4.82 5.22 5.58 4.65
%
3
%
4.80 5.21 5.22 5.58 5.03 5.42
3 % %
3 3 1
5.62 5.62 5.58
z
z
z
4.38 4.36 4.55 4.82 5.22 5.58 4.65
4.17 4.17 4.36 4.39 4.8 I 5.22 4.50
z z
*
z z z
4.58 4.76 4.75 4.82 5.22 5.58 4.90 5.16
4.38 4.56 4.55 4.39 4.81 5.22 4.83 5.03
z z
3
% %
5.21 5.22 5.58
4.80 4.76 4.75 4.82 4.8 1 5.22
3
4.38 4.36 4.55 4.82 5.22 5.58 4.50 5.75
3 3 4.58 4.76 4.75 4.82 5.22 5.58 4.83 4.80
1 3 3 4.80 4.76
% 5.21 5.22 5.58
t 5.08 5.71 4.17 4.17 4.36 4.39 4.8 I 5.22 4.36 5.55
5.72 5.71 4.38 4.56 4.55 4.39 4.8 I 5.22 4.73 4.65 5.75 5.72 5.71 4.58 4.56 4.75 4.82 4.8 I 5.22
Not tested.
t The pH at which growth occurred after incubation for 21 d was the lowest pH tested. 3 A pH higher than that at which no growth occurred was not tested. 4 = acetic acid, 0.3% (w/v). 11 = citric acid, 2.0% (w/v). 7 = lactic acid, 1.0% (w/v). in most cases, to increase the minimum pH that allowed growth. The minimum concentration of organic acids that inhibited growth of Y.enrerocolitica could not be determined reliably where no growth occurred at the highest pH tested. Where growth occurred, however, the calculated minimum concentration of undissociated acetic and lactic acids that inhibited growth after incubation for 21 d was (mmol/l): acetic acid, 2.37 at 10"C, and in the range (depending on strain) 4.9810.68 at 20°C; lactic acid, in the ranges (depending on strain) 4.35485 at 4"C, 4 . 2 6 10.97 at 10°C and 4.17-10.74 at 20°C.
Mscdoa In culture medium acidified with HCI the lowest pH at which Y.enterocolitica multiplied at 20°C in 21 d was pH 4.18, and no growth was detected at pH 3.95 (Table 3). Similar limiting pH values during incubation at 22°C and 25°C have been reported by other workers (Kendall & Gilbert 1980; Brackett 1986). Our results also show the ability of this bacterium to grow in acidic conditions at low temperature; at lo", 7" and 4°C multiplication occurred at pH 4.26, pH 4.36 and pH 4.50 respectively in 21 d. Measurement of the concentration of acetic acid in uninoculated media showed that losses
T . F . Brocklehurst and Barbara M . Lund
394
Table 2. The effect of incubation time, temperature and the presence of organic acids on the minimum pH for growth of Yersinia enterocolitico strain BL 87/46 (serotype 0 : 9) Growth after incubation for (d):
Organic acid 8
Temperature ("C)
TYPe
20
Acetic Citric Lactic
Concn. (mmol/l) 0 17 33 5ori 24 48 9511 22
44 illy 10
Acetic Citric Lactic 7 4
Acetic
0 17 33 50 24 48 95 22
44 Ill 0 0 17 33 50
Citric Lactic
24 48 95 22
44 Ill
14
21
+
-
+
-
+
-
4.28
4.20 5.40
4.20
4.09
5.72
5.51
4.20 5.20 5.72
4.09
5.55
%
5.7 I 4.17 4.36 4.36 4.39 4.81 5.22 4.36 5.75
4.38 4.56 4.55 4.82 5.22 5.58 4.50
% % %
4.58 4.76 4.75 4.82 5.22 5.58
%
5.28
% %
1 % % %
5.21 5.62 5.58
5.72 5.71 4.38 4.56 4.55 4.39 4.81 5.22 5.95 5.16 5.75 5.72 5.7 I 4.80 4.76 4.75 4.82 5.22 5.22
* 4.17 4.36 4.55 4.82 5.22 5.58 4.36
4.38 4.56 4.75 4.82 5.22 5.58
1
4.80
* 4.80 4.76
%
4.82 5.22 5.58
t 4.17 4.36 4.39 4.8 I 5.22 4.28
4.17 4.36 4.55 4.39 4.8 I 5.22 5.95 4.62
*
* 4.58 4.56 4.75 4.39 4.81 5.22
% 4.17 4.36 4.55 4.82 4.8 I 5.22 4.36 5.75
% %
4.38 4.56 4.55
4.82 4.8 I 5.22 4.73 4.62
% % %
4.58 4.76 4.75 4.82 5.22 5.58
t 5.5 I 5.71
t 4.17 4.36 4.39 4.4 I 4.83 4.28 5.55
5.72 5.71 4.17 4.36 4.36 4.39 4.4 I 4.83 4.50 4.50 5.75 5.72 5.71 4.38 4.56 4.55 4.39 4.8 I 5.22
Not tested.
t The pH at which growth occurred after incubation for 21 d was the lowest pH tested. 3 A pH higher than that at which no growth occurred was not tested.
= acetic acid, 0.3% (w/v) 11 =citric acid, 2.0% (w/v). = lactic acid. 1.0% (w/v).
(i
occurred during incubation of microplates. The loss increased with time and was greater in medium adjusted to pH 4.0 than in media adjusted to pH 5.0 or 5.5. It was probably due to loss of the volatile undissociated form of acetic acid. In the work reported here the lowest pH at which growth occurred in the presence of acetic acid was pH 5.20. At this and at higher pH values the loss of acetic acid from microplates incubated at 20°C for 21 d was < 2% of the initial concentration. Lactic acid was not lost throughout incubation of uninoculated microplates at 20°C for 21 d. Citric acid is also a non-volatile acid and
it was assumed that no loss of this acid occurred. The effect of acetic, citric or lactic acid in the medium was, in most cases, to increase the minimum pH for the initiation of growth. In the case of lactic acid this increase was between 0.2 and 0.7 pH unit in the presence of 22 mmol/l lactic acid, between 0.4 and 1.1 pH unit in the presence of 44 mmol/l lactic acid, and between 0.8 and 1.4 pH unit in the presence of I 1 1 mmol/l lactic acid, after incubation at 20°C for 21 d. The effect of acetic acid was more marked, and the increase in the minimum pH for initi-
Initiation of growth of Y.enterocolitica
395
Table 3. The effect of incubation time, temperature and the presence of organic acids on the minimum pH for growth of Yersiniu enterocoliticu strain YE 328 (serotype 0 : 5, 27) Growth after incubation for (d):
Organic acid 8
Temperature ("C)
Type
20
Acetic Citric Lactic
Concn. (mmol/l) 0 17 33 5% 24 48 95 II 22 44
lllq 10
Acetic Citric Lactic 7 4
Acetic Citric Lactic
0 17 33 50 24 48 95 22 44
Ill 0 0 17 33 50 24 48 95 22 44 Ill
14
21
+
-
+
-
+
-
4.26 5.40 5.72
4.18 5.20 5.51 5.71 4.24 4.28 4.48 4.45 4.84 4.83 4.36 5.75 5.72 5.71 4.47 4.48 4.69 4.87 4.84 5.24 4.62 5.18 5.75 5.72 5.71 4.87 4.85 4.88 4.87 5.29 5.24
4.18 5.40 5.72
3.95 5.20 5.51 5.71 4.24 4.28 4.48
4.18 5.20 5.30
3.95
%
4.47 4.48 4.69 4.87 5.29 5.24 4.48
% % t
4.66 4.68 4.88 5.25 5.29 5.66 4.8 I 5.27
% % % % % %
5.25 5.66 5.66
1
4.47 4.48 4.69
* * 4.36 5.75
% %
4.66 4.68 4.69
4.26 5.55 5.72 5.71 4.47 4.48 4.48
* *
4.62 4.92
% % %
4.87 4.85
%
4.48 4.8 1 5.75 5.72 5.71 4.66 4.68 4.88
%
4.47 4.48 4.69 4.87 5.29 5.24 4.26 5.75
% % 4.47 4.68 4.69 4.87 5.29 5.66 4.36 4.62
1
% %
4.87 4.85
%
4.87 5.29 5.66
t 5.08 5.71 4.24 4.28 4.48 4.45 4.84 4.83 4.18 5.55 5.72 5.71 4.24 4.48 4.48 4.45 4.84 5.24 4.26 4.48 5.75 5.72 5.71 4.66 4.68 4.88 4.45 4.84 5.24
* Not tested.
t The pH at which growth occurred after incubation for 21 d was the lowest pH tested. 1A pH higher than that at which no growth occurred was not tested. 8 = acetic acid, 0.3% (w/v) 11 =citric acid, 2.0% (w/v).
T
= lactic acid, 1.0% (w/v).
ation of growth was greater than in the presence of similar total (i.e. undissociated dissociated) concentrations of lactic acid. This increase was between 0.8 and 1.5 pH unit in the presence of 17 mmol/l acetic acid, and between 0.9 and 1.5 pH unit in the presence of 33 mmol/l acetic acid, after incubation at 20°C for 21 d. The effect of citric acid was less marked than that of either acetic or lactic acid. In most cases the minimum pII for the initiation of growth at 20", 10" and 4°C in the presence of 24 mmol/l citric acid was similar to the limiting pH in the absence of citric acid. At the highest concentration tested (95 mmol/l citric acid) an increase in
+
the limiting pH of up to 0.5 unit occurred at 20°C. Growth did not occur in the presence of 50 mmol/l acetic acid at pH 5.71 at any temperature tested, in the presence of 33 mmol/l acetic acid at pH 5.72 and lo" or 4"C, at pH 5.08 or 5.51 and 20°C. and in the presence of 17 mmol/l acetic acid at pH 5.75 and 4"C, and at pH 5.55 and 10°C. Growth was inhibited by lactic acid at a concentration of 22 mmol/l at pH 4.39-4.82, at 4°C. and between pH 4.39 and 4.45 at 10" and 20°C; at a concentration of 44 mmol/l at pH 4.814.84 at 4"C, and at pH 4 . 4 1 4 8 4 at 10"
T . F. Brocklehurst and Barbara M . Lund
396
Table 4. The effect of incubation time. temperature and the presence of organic acids on the minimum pH for growth of Yersinio errrerocolirica strain BL 87/45 (serotype 0 : 8) Organic acid
Growth after incubation for (d): 8
Temperature ("C)
14
21
Type
Concn. (mmol/l)
+
-
+
-
+
-
0 17 33
4.20 5.40 5.72
4.09 5.20 5.51 5.71 4.17 4.36 4.36 4.39 4.81 5.22 4.28 5.75 5.72 5.71 4.17 4.36 4.36 4.39 4.81 5.22 4.50
4.20
4.09
4.20 5.20
4.09
Acetic
5.30
5.08 5.7 1
20
%
5w Citric Lactic
24 48 95 II 22 44
1117 0
10
Acetic Citric Lactic 7 4
Acetic Citric Lactic
17 33 50 24 48 95 22 44 111 0 0 17 33 50 24 48 95 22 44 111
4.38 4.56 4.55 4.82 5.22 5.58 4.36
% % 1
4.38 4.56 4.55 4.82 5.22 5.58 4.65 5.16
% % 1 1 % %
5.2 1 5.22 5.58
5.03 5.75 5.72 5.71 4.80 4.76 4.75 4.82 4.81 5.22
* *
4.17 4.36 4.55 4.82 4.81 5.58 4.28
*
4.38 4.36 4.55 4.82 4.81 5.58 4.50 4.65
4.58 4.56
1
4.82 5.22 5.58
t 4.17 4.36 4.39 4.41 5.22 4.20
4.17 4.17 4.36 4.39 4.41 5.22 4.36 4.50
4.38 4.36 4.75 4.39 4.81 5.22
%
4.17 4.17 4.55 4.82 4.81 5.58 4.28 5.75
% %
4.38 4.36 4.55 4.82 4.81 5.58 4.36 4.50
1 % %
4.38 4.56 4.55 4.82 5.22 5.58
t
t t 4.36 4.39 4.4 1 5.22 4.20 5.55 5.72 5.71 4.17 4.17 4.36 4.39 4.41 5.22 4.28 4.36 5.75 5.72 5.71 4.17 4.36 4.36 4.39 4.8 1 5.22
Not tested.
t The pH at which growth occurred after incubation for 21 d was the lowest pH tested. 1A pH higher than that at which no growth occurred was not tested.
acetic acid. 0.3% (w/v) . . _ icitric acid..2.0% (w/v). 7 i lactic acid, 1.0% (w/v). 6
i
and 20°C; and at a concentration of 11 1 mmol/l at pH 5.22-5.24 at 4"C, at pH 4.83-5.24 at 10°C and at pH 4433-5.22 at 20°C. The antimicrobial activity of weak acids is due mainly to the concentration of undissociated acid (Baird-Parker 1980; Eklund 1985). The concentration of undissociated molecules varies according to the total concentration of acid, the pH and the dissociation constant (pK), which varies with temperature (Robinson & Stokes 1959). In those cases where no growth occurred at the highest pH tested a reliable determination of the inhibitory concentration of undissociated
acid could not be made. If these data are excluded, the calculated inhibitory concentrations of undissociated acetic acid (assuming pK,, = 4.76, pK,, = 4.76 and pK, = 4.77) and lactic acid (assuming pK,, = 3.86, pK,, = 3.87 and pK, = 3.88) were within the ranges found to be lethal to Y. enterocolitica by Brackett (1987). The calculated inhibitory concentrations of undissociated lactic acid were also in agreement with the work of Grau (1981) who found that an 'environmental' strain multiplied in the presence of 3 mmol/l ( = a total concentration of 150 mmol/l at pH 5.55) in culture medium incubated aerobically at 5" or 25°C although this
Initiation of growth of Y.enterocolitica concentration was inhibitory t o cultures incubated anaerobically. W e are grateful t o M r A. Hobson-Frohock a n d Miss Angela Bailey for their collaboration in setting up assays for acetic a n d lactic acids, a n d t o Miss A.J. Thompson for technical assistance. This work was funded by the Ministry of Agriculture. Fisheries a n d Food.
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