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Influence of pH on heat resistance of Bacillus licheniformis in buffer and homogenised foods
International
Journal
of Food Microbiology International Food Microbiology
ELSEVIER
Journal of 29 (1996) l-10
Influence of pH on heat resistance of BuciZZus Zichenifurmis in buffer and homogenised foods A. Palop, J. Raso, R. Pagh, Dpto. P.A.C.A.-Tecnologia
S. Condh,
F.J. Sala *
de 10s Alimentos, Facultad de Veterinaria, Uniuersidad de Zaragoza, Miguel Servet 177, 50.013 Zaragoz, Spain
Received
12 December
1994; accepted
23 January
1995
Abstract The influence of pH of heating menstruum (Mcllvaine buffer) on the heat resistance of licheniformis was investigated and compared with the heat resistance in homogenised tomato and asparagus at pH 7 and 4 in a wide range of temperatures. Heat resistance was in all mestrua smaller at acid pH. At 99°C and pH 4, heat resistance was l/20 lower than at pH 7. However, the magnitude of this effect decreased as heat treatment temperatures were increased almost disappearing at 120°C. z values increased from 6.85 at pH 7, to 10.75 at pH 4. At 99°C the effect of pH on heat resistance was constant along the range of pH’s tested. The increase of one pH unit increased D,, by 180%. At pH 7 and 4, heat resistance was the same in buffer as in tomato and asparagus homogenates at all temperatures tested. The diminishing influence of the acidification of some foods on the heat resistance of B. licheniformis sterilisation temperatures should be taken into account when a raise in temperature is considered to shorten the duration of heat processes.
Bacillus
Keywords:
Sterilisation;
Bacillus
licheniformis;
Heat resistance; pH
1. Introduction One of the attempts being made to avoid unwanted effects of heat treatments on foods is through the combined use of different preservation methods. This procedure is currently known as Preservation of Foods by Combined Processes. The combination of heat and acid pH’s was one of the first Combined Processes to be used, with the objective of reducing the intensity of heat treatments of some
* Corresponding
author.
Tel.: 76-761581.
0168-1605/96/$15.00 0 1996 Elsevier SSDI 0168-1605(95)00010-0
Fax: 761612.
Science
B.V. All rights reserved
2
A. Palop et al. /ht.
J. Food Microbiology 29 (1996) l-10
canned vegetables. Today, the acidification of some canned vegetable to pH’s lower than 4.5 is a normal practice. This practice has the advantage of reducing heat resistance of some heat-resistant microorganisms (usual contaminators of these products), but also of preventing the germination of survivors and of spores of Clostridium botulinum escaping heat treatment. Bacillus licheniformis is one of microorganisms most frequently involved in the spoilage of canned vegetables (Fields et al., 1977). Its ability to grow in very acidic media and its reported capability of neutralising its growth medium to pH’s in which C. botulinum would be able to grow and produce its toxin (Fields et al., 1977; Rodriguez et al., 1993), has made it important in acidified canned vegetables as its survival from heat processes may increase the risk of botulinic intoxication. It has been long known that pH of heat treatment medium influences heat resistance of bacteria. Most authors agree that heat resistance is biggest at pH close to neutrality decreasing there from, with acidification (Xezones and Hutchings, 1965; Lowick and Anema, 1972; Mazokhina et al., 1973; Brown and Thorpe, 1978; Cameron et al., 1980; Cerny, 1980). The magnitude of this effect and the range of pH’s where this is biggest seems to vary, not only among species (Cerny, 1980) but also among different strains of the same species (Brown and Thorpe, 1978). But the influence of pH of heat treatment medium on z values is a very important matter still not clear. While some authors (Brown and Thorpe, 1978; Cerny, 1980) reported an increase of z value with acidification, others found that this effect was reversed (Cameron et al., 1980) and still some investigators (Lowick and Anema, 1972) reported that pH of medium had no influence. On the other hand, some effect of the composition of menstruum on heat resistance has also been reported (Condon and Sala, 1992). Most authors advise that heat resistance experiments are carried out in the same food to be processed (Stumbo, 1965; Cameron et al., 1980; Pflug, 1987). Despite the possible important role of B. licheniformis in the increase of the risk for an eventual botulism outbreak in canned vegetables, data available on its heat resistance are very scarce and it has been obtained in very different methodological conditions. Furthermore, the influence on its heat resistance of different environmental factors is absolutely unknown. The objective of this investigation was to determine the influence of pH and composition of menstruum during heat treatment on the heat resistance of B. ficheniformis, in a wide range of temperatures of treatment (z values) with the purpose of assessing the adequacy of current acidification and heat-processing practices. 2. Materials
and methods
2.1. Microorganims The strain of B. licheniformis used in this investigation Collection no. 4523) was isolated in the laboratories
(Spanish of AICV
Type Culture (San Adrian,
A. Palop et al. /ht.
J. Food Microbiology
29 (1996) I-10
3
Navarra, Spain) during a routine control for sterility of canned asparagus and was identified in our laboratory by the API Identification System (50 CHB) and the Voges-Proskauer, nitrate reduction, citrate and propionate utilization test. Sporulation was carried out in Roux flasks of nutrient agar (Biolife, Milano, Italy) with 500 mg/l of Bacto Dextrose (Difco, Michigan, USA) and 3 mg/l of manganese sulphate (Probus, Barcelona, Spain). Flasks were inoculated with a young culture (24 h at 35°C) in nutrient broth (Biolife) and incubated during 5 days at 35°C. After that time 80% of sporulation was attained. Spores were collected from the agar surface with sterile McIlvaine buffer (pH 7) and glass beads (Dawson et al., 1974). The suspension obtained was washed five times by centrifugation and resuspension in this same buffer. The final suspension, having a concentration of 107-lo8 spores/ml, was stored under refrigeration (0-5°C) until used. During storage no variations in heat resistance were observed. 2.2. Heat treatments Heat treatments were carried out in a TR-SC thermoresistometer as described by Condon et al. (1989). Once heat treatment temperature had attained stability, the menstruum was inoculated with 0.2 ml of the spores suspension. At preset intervals, 0.1 ml samples were directly collected into tubes of melted sterile nutrient agar (Biolife) containing 500 mg/l of Bacto Dextrose (Difco), which were immediately plated. 2.3. Menstruum McIlvaine buffers (pH 4, 5, 6 and 7) were prepared as described by Dawson et al. (1974) and were stored under refrigeration (0-5°C) until used. Asparagus were prepared as usually done in canning factories (Conservas Barcos, Caparroso, Navarra, Spain). However, in this case no acidulants were added before sterilisation. After preparation, the product was stored frozen until used. For heat-resistant experiments it was homogenised and filtered through a sterile cloth. Tomato was supplied frozen by a canning factory (Congelados Virtos, Cortes, Navarra, Spain) and was prepared as asparagus. Acidification of menstruum was done in the thermoresistometer once the stability of heat treatment temperature was attained. Acidifications were done with hydrochloric acid to pH 4 and alkalinizations with 1 N sodium hydroxide to pH 7. 2.4. Incubation and survival counting Plates were incubated during 24 h at 35°C. Previous experiments showed that longer incubation times did not modify significantly the profile of survival curves. Survival counting of plates with a high density of colony forming units (CFU) was done as previously described (Condon et al., 1987).
4
A. Palop et al. /Int. J. Food Microbiology 29 (1996) l-10
2.5. D, and z values D, values were calculated from the straight portion of survival curves (obtained by plotting log of survival counts vs. their corresponding heating times). Only survival curves with more than four values in the straight portion, with a coefficient of correlation (r,) 2 0.97, and descending more than one log cycle were used. z values were determined from the regression line obtained by plotting log D, values vs. their corresponding heating temperatures (Decimal Reduction Time Curve; DRTC). Comparison of slopes of survival curves and DRTC was performed as it has been described by Steel and Torrie (1960). Coefficients of correlation (r,) and 95% confidence limits (CL) were calculated by the appropriate statistical package (Statview 512; D. Feldman and J. Gagnon, Brain Power Inc., Calabasas, CA, USA). 3. Results The precision of results values, their corresponding
obtained is shown in Tables l-4. Table 1 includes D, 95% CL and the r, values of survival curves obtained
Table 1 Influence of pH of the heating menstruum (Mcllvaine buffer) upon the heat resistance (D, values) of B. licheniformis PH
T (“C)
D, (mid
95%-
CL
95%+
CL
4
92.9 96.0 99.0 102.0 108.0 113.9
0.48 0.26 0.23 0.081 0.031 0.0048
0.42 0.22 0.19 0.070 0.029 0.0036
0.56 0.32 0.29 0.096 0.034 0.0074
0.997 0.998 0.993 0.996 0.998 0.983
5
93.0 99.0 104.9 111.0
2.3 0.52 0.093 0.019
1.9 0.44 0.061 0.017
2.9 0.63 0.197 0.020
0.994 0.991 0.985 0.998
99.0 105.0 110.8 116.9
1.5 0.17 0.038 0.0055
1.4 0.13 0.032 0.0042
1.7 0.26 0.046 0.0077
0.995 0.994 0.981 0.979
99.0 102.0 111.0 113.9 119.9
4.2 2.2 0.11 0.033 0.0042
3.7 2.1 0.10 0.029 0.0034
4.8 2.4 0.13 0.038 0.0054
0.994 0.999 0.998 0.994 0.997
rn
A. Palop et al. /ht.
J. Food Microbiology
29 (1996) I-10
5
Table 2 Influence of pH of the heating menstruum (McIlvaine buffer) upon the z values of B. licheniformis PH
z
95%-
4 5 6 7
10.75 8.54 7.46 6.85
8.70 7.87 6.29 6.21
CL
95%+
CL
r0
13.88 9.43 9.26 7.69
0.987 0.999 0.998 0.998
at different heating temperatures in buffer of pH 4, 5, 6 and 7. Table 2 includes z values with their 95% CL and T,, values of corresponding DRTC. Table 3 include D, values with their 95% CL and rO values of survival curves obtained at different temperatures with homogenised tomato and asparagus at pH 7 and 4. Table 4 includes z values, their 95% CL and r. values of corresponding DRTC curves. Coefficients of correlation and 95% CL included in these tables are among the
Table 3 Influence of pH of the heating menstruum upon the heat resistance (D, values) of B. licheniformis tomato and asparagus Menstruum
T (“Cl
D, (min)
95%-
Tomato pH 4
93.0 96.0 102.0 104.8 107.9 110.9 116.9
0.48 0.25 0.067 0.036 0.019 0.011 0.0025
0.41 0.19 0.063 0.029 0.017 0.010 0.0023
CL
0.58 0.36 0.072 0.048 0.022 0.012 0.0028
95%+
CL
0.998 0.995 0.999 0.997 0.997 0.996 0.997
Tomato pH 7
99.0 105.0 111.0 116.9
3.6 0.60 0.12 0.0092
3.2 0.51 0.09 0.0073
4.0 0.75 0.16 0.0126
0.993 0.994 0.996 0.996
Asparagus pH 4
86.9 90.0 93.2 99.0 102.1 110.9 117.0
1.0 0.61 0.30 0.11 0.10 0.011 0.0021
1.0 0.411 0.24 0.10 0.09 0.010 0.0013
1.2 1.22 0.39 0.14 0.12 0.012 0.0057
0.996 0.987 0.997 0.993 0.997 0.999 0.979
Asparagus pH 7
99.1 102.2 108.1 111.0 116.8
3.3 1.2 0.20 0.10 0.0090
2.4 0.9 0.16 0.09 0.0077
5.5 1.5 0.27 0.11 0.0108
0.992 0.984 0.991 0.996 0.983
r0
in
6 Table 4 Influence asparagus
A. Paiop et (11./ Int. J. Food Microbiology
of pH of the heating
menstruum
upon
the
29 (1996) 1 -IO
z values
of B. licheniformis
in tomato
Menstruum
PH
.?
950/F-
Tomato
4 7
10.64 7.04
10.20 5.26
10.99 10.64
0.999 0.994
Asparagus
4 7
11.49 7.09
10.10 6.13
13.51 8.40
0.992 0.996
CL
95%+
CL
and
rll
best found in the literature (Pflug and Odlaug, 1978; Mikolajcik and Rajkowski, 1980). Fig. 1A shows survival curves of B. licheniformis obtained at 99°C in buffers of pH 4, 5, 6 and 7 and Fig. 1B the relationship between the log of the estimated D,, values vs. pH of heat treatment medium. As seen in this figure, this was an exponential-like relationship.
Fig. 1. Influence of pH of heating menstruum (McIlvaine buffer) on the heat resistance of B. licheniformis. (A) Survival curves at 99°C of B. licheniformis at pH 4 f + ), pH 5 (A), pH 6 (0) and pH 7 to different pH’s of the heating menstruum. (0). (B) D,, values corresponding
A. Palop et al. /Int. J. Food Microbiology 29 (1996) I-10
7
Temperature (“C)
Fig. 2. Thermal
death
time curves of B. licheniformis in McIlvaine
buffer
of pH 4 (+ ), pH 5 (A), pH 6
(0) and pH 7 (@I.
The magnitude of the effect the temperature of treatment. buffers of pH 4, 5, 6 and 7. As pH 7 to pH 4 caused z values
of pH of medium on heat resistance depended on Fig. 2 shows DRTC of B. Zicheniformis obtained in seen in this figure, the acidification of medium from to increase from 6.85 to 10.75.
0A
-3 90
95
loo
105 Temperamre
110
120
125
(“C)
0B
Tempemure
(‘C)
Fig. 3. Thermal death time curves of B. licheniformis in foods and buffers. (A) TDT curves in McIlvaine buffer of pH 4 (+) and 7 (0) and tomato at pH 4 (0) and 7 (A). (B) TDT curves in Mcllvaine buffer of pH 4 (+I and 7 (0) and asparagus at pH 4 (0) and 7 (A).
8
A. Palop et al. /ht.
J. Food Microbiology 29 (1996) 1 -IO
In Fig. 3A, DRTC in homogenised tomato after adjusting pH to 7 and 4 are shown. DRTC obtained in buffers of pH 4 and 7 have been included in this figure as a reference. Fig. 3B includes DRTC obtained in homogenised asparagus, at pH 7 and 4, and in buffer at these same pH’s.
4. Discussion The pH of heat treatment medium had a strong influence on the heat resistance of our strain of B. licheniformis (Table 1). Heat resistance at acid pH’s was lower. Our results are in agreement with most published data on different BacilZus spp. (Mazokhina et al., 1973; Leitao et al., 1977; Brown and Thorpe, 1978; Cerny, 1980; Nakajo and Moriyama, 1993). In our strain, the magnitude of this effect depended on the temperature of treatment. The effect observed at lower sterilisation temperatures was much bigger than those reported in the literature for different Bacdlus spp. while the average reduction of heat resistance from pH 7 to 4 reported by authors is by l/4 to l/6 (Leitao et al., 1977; Cerny, 1980) with our strain, the reduction of D,, value was by l/20 (Fig. 1A). This reduction was much bigger than the l/2 reduction reported by Montville and Sapers (1981) for a B. ficheniformis strain in tomato in the same range of pH’s. Perhaps this disagreement could be due to a missidentification as reported by Rodriguez et al. (1993). The relationship between log D,, and pH of menstruum during heat treatment in this pH range (pH 4-7) seemed to be linear (Fig. 1B). One unit increment in pH increased D,, by approximately 180%. The behaviour of our strain is different from that reported for most of Bacillus spp. in which the acidification from pH 7 to pH 6 almost had no influence on heat resistance (Leitao et al., 1977; Brown and Thorpe, 1978; Montville and Sapers, 1981). A similar behaviour than that of our strain has been reported for C. sporogenes and B. stearothennophilus (Mazokhina et al., 1973). Authors disagree on the influence of pH on z values as mentioned earlier. These disagreements could be due to actual differences in behaviour among different species or to different sporulation conditions as observed by some authors (Sala et al., 1995). The decrease in heat resistance of our strain at acid pH’s became smaller the higher the temperature of treatment (Fig. 2). .z = 6.8 at pH 7 increased to z = 10.7 at pH 4 (Table 2). Our z values were smaller that those reported by Montville and Sapers (1981) for two B. licheniformis strains in tomato of pH 4.4 (z = 14.5) and by Rodriguez et al. (1993) in the same menstruum (z = 14.2) but similar to those reported by Behringer and Kessler (1992) in milk (z = 7.5-8’0. There seems to be no information published on the influence of pH on z values of B. licheniformis with which our results can be compared directly. The influence of the composition of heating menstruum on heat resistance reported by some authors may be due, in some authors opinion (Brown and Thorpe, 19781, not to medium composition itself, but to differences in pH and water activity (a,).
A. Palop et al. / Int. J. Food Microbiology
29 (1996) I-10
9
According to our results, the influence of menstruum in the heat resistance of our strain is mainly due to pH (Table 3). The heat resistance in tomato and asparagus at pH 7 and 4 (Fig. 3A,B) was practically the same as that in McIlvaine buffer of these same pH’s. The effect of pH of food, as it happened with buffer, also decreased at higher temperatures of treatment and no statistically significant differences (p I 0.011 were detected either between .z values obtained with foods and with buffer at the same pH (Tables 2 and 4). However, in former investigations (Condon and Sala, 19921, it was observed that heat resistance of B. subtilis in food at its natural pH was somewhat different than that in buffer of the same pH. Maybe actual differences in behaviour among species or sporulation conditions could account for these differences. Remarkable influences of sporulation temperature on D, and z values of B. subtilis have been reported (Sala et al., 1995). No general conclusions can be drawn on the influence of pH of foods on z value, given the diversity of behaviours reported by investigators of the various species studied in different heating media. The acidification of heating menstruum, regardless of its composition, decreased heat resistance of our strain of B. Zicheniformis. But the magnitude of this decrease was smaller with increasing temperature being almost nil at the highest temperatures of treatment investigated.
Acknowledgements
This study was supported in part by Diputacion General de Aragon (Project P CA-5/89) which also provided a grant to A. Palop to carry out this investigation.
References Behringer, R. and Kessler, H.G. (1992) Influence of pH value and concentration of skimmed milk on heat resistance of Bacillus licheniformis and B. stearothermophilm spores. Milchwissenschaft 47, 207-211. Brown, K.L. and Thorpe, R.H. (1978) The relationship between pH and the heat resistance and recovery of bacterial spores. C.F.P.R.A. Tech. Mem. n1204. Cameron, M.S., Leonard, S.J. and Barret, E.L. (1980) Effect of moderately acidic pH on heat resistance of Clostridium sporogenes spores in phosphate buffer and in buffered pea puree. Appl. Environ. Microbial. 39, 943-949. Cerny, G. (1980) Dependence of thermal inactivation of microorganisms on the pH-value of media. II. Bacteria and bacterial spores. Z. Lebensm. Unters. Forsch. 170, 180-186. Condon, S. and Sala, F.J. (1992) Heat resistance of Bacillus subtilis in buffer and foods of different pH. J. Food Protect. 55, 605-608. Condon, S., Oria, R. and Sala, F.J. (1987) Heat resistance of microorganisms: An improved method for survival counting. J. Microbial. Methods 7, 37-44. Condon, S., Lopez, P., Oria, R. and Sala, F.J. (1989) Thermal death determination: design and evaluation of a thermoresistometer. J. Food Sci. 54, 451-457.
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A. Paiop et al. /Int. J. Food Microbiology 29 (1996) I-IO
Dawson, R.M.C., Elliot, D.C., Elliot, W.H. ano Jones, K.M. (1974) Data for Biochemical Research. Oxford at the Clarendon Press, Oxford, pp. 484-485. Fields, M.L., Zamora, A.F. and Bradsher, M. (1977) Microbiological analysis of home-canned tomatoes and green beans. J. Food Sci. 42, 931-934. Leitao, M.F.F., Ordorico, CA., Ciampi, C. and Quat, D.G. (1977) The thermal resistance of Bacillus stearothermophilus and Clostridium PA 3679 spores in banana puree. Cal. Inst. Technol. Aliment. 8. 313-327. Lowick, J.A.M. and Anema, P.J. (1972) Effect of pH on the heat resistance of Cl. sporogenes spores in minced meat. J. Appl. Bacterial. 35, 1199121. Mazokhina, N.N., Naidenova, L.P., Rozanova, L.1. and Dashevskaya, T.V. (1973) Heat resistance and pH effect on microorganisms, causing spoilage of canned foods. Acta Aliment. 2, 385-391. Mikolajcik, E.M. and Rajkowski, K.T. (1980) Simple technique to determine heat resistance of Bacillus stearothermophilus spores in fluid systems. J. Food Protect. 43. 799-804. Montville, T.J. and Sapers, G.M. (1981) Thermal resistance of spores from pH elevating strains of Bacillus licheniformis. J. Food Sci. 46, 1710-1712, 1715. Nakajo, M. and Moriyama, Y. (1993) Effect of pH and water activity on heat resistance of Bacillus coagulans spores. J. Jpn. Sot. Food Sci. Technol. 40, 268-271. Pflug, I.J. (19871 Factors important in determining the heat process value, F,, for low-acid canned foods. J. Food Protect. 50, 528-533. Pflug, I.J. and Odlaug, T.E. (1978) A review of Z and F values used to ensure the safety of low-acid canned foods. Food Technol. 32, 63-70. Rodriguez, J.H., Cousin, M.A. and Nelson, P.E. (1993) Thermal resistance and growth of Bacillus licheniformis and Bacillus subtilis in tomato juice. J. Food Protect. 56. 1655168. Sala, F.J., Ibarz, P., Palop, A., Raso, J. and Condon, S. (1995) Sporulation temperature and heat resistance of Bacillus subtilis at different pH’s. J. Food Protect. 58, 239-263. Steel, R.G. and Torrie, J.H. (19601 Principles and Procedures of Statistics. McGraw Hill Book Co. Inc., New York, pp. 161-180. Stumbo, CR. (1965) Thermobacteriology in Food Processing. Academic Press Inc., New York, pp. 98-102. Xezones, H. and Hutchings, I.J. (1965) Thermal resistance of Clostridium botulinum (62A) spores as affected by fundamental food constituents. Food Technol. 19, 113-l 15.
Journal
of Food Microbiology International Food Microbiology
ELSEVIER
Journal of 29 (1996) l-10
Influence of pH on heat resistance of BuciZZus Zichenifurmis in buffer and homogenised foods A. Palop, J. Raso, R. Pagh, Dpto. P.A.C.A.-Tecnologia
S. Condh,
F.J. Sala *
de 10s Alimentos, Facultad de Veterinaria, Uniuersidad de Zaragoza, Miguel Servet 177, 50.013 Zaragoz, Spain
Received
12 December
1994; accepted
23 January
1995
Abstract The influence of pH of heating menstruum (Mcllvaine buffer) on the heat resistance of licheniformis was investigated and compared with the heat resistance in homogenised tomato and asparagus at pH 7 and 4 in a wide range of temperatures. Heat resistance was in all mestrua smaller at acid pH. At 99°C and pH 4, heat resistance was l/20 lower than at pH 7. However, the magnitude of this effect decreased as heat treatment temperatures were increased almost disappearing at 120°C. z values increased from 6.85 at pH 7, to 10.75 at pH 4. At 99°C the effect of pH on heat resistance was constant along the range of pH’s tested. The increase of one pH unit increased D,, by 180%. At pH 7 and 4, heat resistance was the same in buffer as in tomato and asparagus homogenates at all temperatures tested. The diminishing influence of the acidification of some foods on the heat resistance of B. licheniformis sterilisation temperatures should be taken into account when a raise in temperature is considered to shorten the duration of heat processes.
Bacillus
Keywords:
Sterilisation;
Bacillus
licheniformis;
Heat resistance; pH
1. Introduction One of the attempts being made to avoid unwanted effects of heat treatments on foods is through the combined use of different preservation methods. This procedure is currently known as Preservation of Foods by Combined Processes. The combination of heat and acid pH’s was one of the first Combined Processes to be used, with the objective of reducing the intensity of heat treatments of some
* Corresponding
author.
Tel.: 76-761581.
0168-1605/96/$15.00 0 1996 Elsevier SSDI 0168-1605(95)00010-0
Fax: 761612.
Science
B.V. All rights reserved
2
A. Palop et al. /ht.
J. Food Microbiology 29 (1996) l-10
canned vegetables. Today, the acidification of some canned vegetable to pH’s lower than 4.5 is a normal practice. This practice has the advantage of reducing heat resistance of some heat-resistant microorganisms (usual contaminators of these products), but also of preventing the germination of survivors and of spores of Clostridium botulinum escaping heat treatment. Bacillus licheniformis is one of microorganisms most frequently involved in the spoilage of canned vegetables (Fields et al., 1977). Its ability to grow in very acidic media and its reported capability of neutralising its growth medium to pH’s in which C. botulinum would be able to grow and produce its toxin (Fields et al., 1977; Rodriguez et al., 1993), has made it important in acidified canned vegetables as its survival from heat processes may increase the risk of botulinic intoxication. It has been long known that pH of heat treatment medium influences heat resistance of bacteria. Most authors agree that heat resistance is biggest at pH close to neutrality decreasing there from, with acidification (Xezones and Hutchings, 1965; Lowick and Anema, 1972; Mazokhina et al., 1973; Brown and Thorpe, 1978; Cameron et al., 1980; Cerny, 1980). The magnitude of this effect and the range of pH’s where this is biggest seems to vary, not only among species (Cerny, 1980) but also among different strains of the same species (Brown and Thorpe, 1978). But the influence of pH of heat treatment medium on z values is a very important matter still not clear. While some authors (Brown and Thorpe, 1978; Cerny, 1980) reported an increase of z value with acidification, others found that this effect was reversed (Cameron et al., 1980) and still some investigators (Lowick and Anema, 1972) reported that pH of medium had no influence. On the other hand, some effect of the composition of menstruum on heat resistance has also been reported (Condon and Sala, 1992). Most authors advise that heat resistance experiments are carried out in the same food to be processed (Stumbo, 1965; Cameron et al., 1980; Pflug, 1987). Despite the possible important role of B. licheniformis in the increase of the risk for an eventual botulism outbreak in canned vegetables, data available on its heat resistance are very scarce and it has been obtained in very different methodological conditions. Furthermore, the influence on its heat resistance of different environmental factors is absolutely unknown. The objective of this investigation was to determine the influence of pH and composition of menstruum during heat treatment on the heat resistance of B. ficheniformis, in a wide range of temperatures of treatment (z values) with the purpose of assessing the adequacy of current acidification and heat-processing practices. 2. Materials
and methods
2.1. Microorganims The strain of B. licheniformis used in this investigation Collection no. 4523) was isolated in the laboratories
(Spanish of AICV
Type Culture (San Adrian,
A. Palop et al. /ht.
J. Food Microbiology
29 (1996) I-10
3
Navarra, Spain) during a routine control for sterility of canned asparagus and was identified in our laboratory by the API Identification System (50 CHB) and the Voges-Proskauer, nitrate reduction, citrate and propionate utilization test. Sporulation was carried out in Roux flasks of nutrient agar (Biolife, Milano, Italy) with 500 mg/l of Bacto Dextrose (Difco, Michigan, USA) and 3 mg/l of manganese sulphate (Probus, Barcelona, Spain). Flasks were inoculated with a young culture (24 h at 35°C) in nutrient broth (Biolife) and incubated during 5 days at 35°C. After that time 80% of sporulation was attained. Spores were collected from the agar surface with sterile McIlvaine buffer (pH 7) and glass beads (Dawson et al., 1974). The suspension obtained was washed five times by centrifugation and resuspension in this same buffer. The final suspension, having a concentration of 107-lo8 spores/ml, was stored under refrigeration (0-5°C) until used. During storage no variations in heat resistance were observed. 2.2. Heat treatments Heat treatments were carried out in a TR-SC thermoresistometer as described by Condon et al. (1989). Once heat treatment temperature had attained stability, the menstruum was inoculated with 0.2 ml of the spores suspension. At preset intervals, 0.1 ml samples were directly collected into tubes of melted sterile nutrient agar (Biolife) containing 500 mg/l of Bacto Dextrose (Difco), which were immediately plated. 2.3. Menstruum McIlvaine buffers (pH 4, 5, 6 and 7) were prepared as described by Dawson et al. (1974) and were stored under refrigeration (0-5°C) until used. Asparagus were prepared as usually done in canning factories (Conservas Barcos, Caparroso, Navarra, Spain). However, in this case no acidulants were added before sterilisation. After preparation, the product was stored frozen until used. For heat-resistant experiments it was homogenised and filtered through a sterile cloth. Tomato was supplied frozen by a canning factory (Congelados Virtos, Cortes, Navarra, Spain) and was prepared as asparagus. Acidification of menstruum was done in the thermoresistometer once the stability of heat treatment temperature was attained. Acidifications were done with hydrochloric acid to pH 4 and alkalinizations with 1 N sodium hydroxide to pH 7. 2.4. Incubation and survival counting Plates were incubated during 24 h at 35°C. Previous experiments showed that longer incubation times did not modify significantly the profile of survival curves. Survival counting of plates with a high density of colony forming units (CFU) was done as previously described (Condon et al., 1987).
4
A. Palop et al. /Int. J. Food Microbiology 29 (1996) l-10
2.5. D, and z values D, values were calculated from the straight portion of survival curves (obtained by plotting log of survival counts vs. their corresponding heating times). Only survival curves with more than four values in the straight portion, with a coefficient of correlation (r,) 2 0.97, and descending more than one log cycle were used. z values were determined from the regression line obtained by plotting log D, values vs. their corresponding heating temperatures (Decimal Reduction Time Curve; DRTC). Comparison of slopes of survival curves and DRTC was performed as it has been described by Steel and Torrie (1960). Coefficients of correlation (r,) and 95% confidence limits (CL) were calculated by the appropriate statistical package (Statview 512; D. Feldman and J. Gagnon, Brain Power Inc., Calabasas, CA, USA). 3. Results The precision of results values, their corresponding
obtained is shown in Tables l-4. Table 1 includes D, 95% CL and the r, values of survival curves obtained
Table 1 Influence of pH of the heating menstruum (Mcllvaine buffer) upon the heat resistance (D, values) of B. licheniformis PH
T (“C)
D, (mid
95%-
CL
95%+
CL
4
92.9 96.0 99.0 102.0 108.0 113.9
0.48 0.26 0.23 0.081 0.031 0.0048
0.42 0.22 0.19 0.070 0.029 0.0036
0.56 0.32 0.29 0.096 0.034 0.0074
0.997 0.998 0.993 0.996 0.998 0.983
5
93.0 99.0 104.9 111.0
2.3 0.52 0.093 0.019
1.9 0.44 0.061 0.017
2.9 0.63 0.197 0.020
0.994 0.991 0.985 0.998
99.0 105.0 110.8 116.9
1.5 0.17 0.038 0.0055
1.4 0.13 0.032 0.0042
1.7 0.26 0.046 0.0077
0.995 0.994 0.981 0.979
99.0 102.0 111.0 113.9 119.9
4.2 2.2 0.11 0.033 0.0042
3.7 2.1 0.10 0.029 0.0034
4.8 2.4 0.13 0.038 0.0054
0.994 0.999 0.998 0.994 0.997
rn
A. Palop et al. /ht.
J. Food Microbiology
29 (1996) I-10
5
Table 2 Influence of pH of the heating menstruum (McIlvaine buffer) upon the z values of B. licheniformis PH
z
95%-
4 5 6 7
10.75 8.54 7.46 6.85
8.70 7.87 6.29 6.21
CL
95%+
CL
r0
13.88 9.43 9.26 7.69
0.987 0.999 0.998 0.998
at different heating temperatures in buffer of pH 4, 5, 6 and 7. Table 2 includes z values with their 95% CL and T,, values of corresponding DRTC. Table 3 include D, values with their 95% CL and rO values of survival curves obtained at different temperatures with homogenised tomato and asparagus at pH 7 and 4. Table 4 includes z values, their 95% CL and r. values of corresponding DRTC curves. Coefficients of correlation and 95% CL included in these tables are among the
Table 3 Influence of pH of the heating menstruum upon the heat resistance (D, values) of B. licheniformis tomato and asparagus Menstruum
T (“Cl
D, (min)
95%-
Tomato pH 4
93.0 96.0 102.0 104.8 107.9 110.9 116.9
0.48 0.25 0.067 0.036 0.019 0.011 0.0025
0.41 0.19 0.063 0.029 0.017 0.010 0.0023
CL
0.58 0.36 0.072 0.048 0.022 0.012 0.0028
95%+
CL
0.998 0.995 0.999 0.997 0.997 0.996 0.997
Tomato pH 7
99.0 105.0 111.0 116.9
3.6 0.60 0.12 0.0092
3.2 0.51 0.09 0.0073
4.0 0.75 0.16 0.0126
0.993 0.994 0.996 0.996
Asparagus pH 4
86.9 90.0 93.2 99.0 102.1 110.9 117.0
1.0 0.61 0.30 0.11 0.10 0.011 0.0021
1.0 0.411 0.24 0.10 0.09 0.010 0.0013
1.2 1.22 0.39 0.14 0.12 0.012 0.0057
0.996 0.987 0.997 0.993 0.997 0.999 0.979
Asparagus pH 7
99.1 102.2 108.1 111.0 116.8
3.3 1.2 0.20 0.10 0.0090
2.4 0.9 0.16 0.09 0.0077
5.5 1.5 0.27 0.11 0.0108
0.992 0.984 0.991 0.996 0.983
r0
in
6 Table 4 Influence asparagus
A. Paiop et (11./ Int. J. Food Microbiology
of pH of the heating
menstruum
upon
the
29 (1996) 1 -IO
z values
of B. licheniformis
in tomato
Menstruum
PH
.?
950/F-
Tomato
4 7
10.64 7.04
10.20 5.26
10.99 10.64
0.999 0.994
Asparagus
4 7
11.49 7.09
10.10 6.13
13.51 8.40
0.992 0.996
CL
95%+
CL
and
rll
best found in the literature (Pflug and Odlaug, 1978; Mikolajcik and Rajkowski, 1980). Fig. 1A shows survival curves of B. licheniformis obtained at 99°C in buffers of pH 4, 5, 6 and 7 and Fig. 1B the relationship between the log of the estimated D,, values vs. pH of heat treatment medium. As seen in this figure, this was an exponential-like relationship.
Fig. 1. Influence of pH of heating menstruum (McIlvaine buffer) on the heat resistance of B. licheniformis. (A) Survival curves at 99°C of B. licheniformis at pH 4 f + ), pH 5 (A), pH 6 (0) and pH 7 to different pH’s of the heating menstruum. (0). (B) D,, values corresponding
A. Palop et al. /Int. J. Food Microbiology 29 (1996) I-10
7
Temperature (“C)
Fig. 2. Thermal
death
time curves of B. licheniformis in McIlvaine
buffer
of pH 4 (+ ), pH 5 (A), pH 6
(0) and pH 7 (@I.
The magnitude of the effect the temperature of treatment. buffers of pH 4, 5, 6 and 7. As pH 7 to pH 4 caused z values
of pH of medium on heat resistance depended on Fig. 2 shows DRTC of B. Zicheniformis obtained in seen in this figure, the acidification of medium from to increase from 6.85 to 10.75.
0A
-3 90
95
loo
105 Temperamre
110
120
125
(“C)
0B
Tempemure
(‘C)
Fig. 3. Thermal death time curves of B. licheniformis in foods and buffers. (A) TDT curves in McIlvaine buffer of pH 4 (+) and 7 (0) and tomato at pH 4 (0) and 7 (A). (B) TDT curves in Mcllvaine buffer of pH 4 (+I and 7 (0) and asparagus at pH 4 (0) and 7 (A).
8
A. Palop et al. /ht.
J. Food Microbiology 29 (1996) 1 -IO
In Fig. 3A, DRTC in homogenised tomato after adjusting pH to 7 and 4 are shown. DRTC obtained in buffers of pH 4 and 7 have been included in this figure as a reference. Fig. 3B includes DRTC obtained in homogenised asparagus, at pH 7 and 4, and in buffer at these same pH’s.
4. Discussion The pH of heat treatment medium had a strong influence on the heat resistance of our strain of B. licheniformis (Table 1). Heat resistance at acid pH’s was lower. Our results are in agreement with most published data on different BacilZus spp. (Mazokhina et al., 1973; Leitao et al., 1977; Brown and Thorpe, 1978; Cerny, 1980; Nakajo and Moriyama, 1993). In our strain, the magnitude of this effect depended on the temperature of treatment. The effect observed at lower sterilisation temperatures was much bigger than those reported in the literature for different Bacdlus spp. while the average reduction of heat resistance from pH 7 to 4 reported by authors is by l/4 to l/6 (Leitao et al., 1977; Cerny, 1980) with our strain, the reduction of D,, value was by l/20 (Fig. 1A). This reduction was much bigger than the l/2 reduction reported by Montville and Sapers (1981) for a B. ficheniformis strain in tomato in the same range of pH’s. Perhaps this disagreement could be due to a missidentification as reported by Rodriguez et al. (1993). The relationship between log D,, and pH of menstruum during heat treatment in this pH range (pH 4-7) seemed to be linear (Fig. 1B). One unit increment in pH increased D,, by approximately 180%. The behaviour of our strain is different from that reported for most of Bacillus spp. in which the acidification from pH 7 to pH 6 almost had no influence on heat resistance (Leitao et al., 1977; Brown and Thorpe, 1978; Montville and Sapers, 1981). A similar behaviour than that of our strain has been reported for C. sporogenes and B. stearothennophilus (Mazokhina et al., 1973). Authors disagree on the influence of pH on z values as mentioned earlier. These disagreements could be due to actual differences in behaviour among different species or to different sporulation conditions as observed by some authors (Sala et al., 1995). The decrease in heat resistance of our strain at acid pH’s became smaller the higher the temperature of treatment (Fig. 2). .z = 6.8 at pH 7 increased to z = 10.7 at pH 4 (Table 2). Our z values were smaller that those reported by Montville and Sapers (1981) for two B. licheniformis strains in tomato of pH 4.4 (z = 14.5) and by Rodriguez et al. (1993) in the same menstruum (z = 14.2) but similar to those reported by Behringer and Kessler (1992) in milk (z = 7.5-8’0. There seems to be no information published on the influence of pH on z values of B. licheniformis with which our results can be compared directly. The influence of the composition of heating menstruum on heat resistance reported by some authors may be due, in some authors opinion (Brown and Thorpe, 19781, not to medium composition itself, but to differences in pH and water activity (a,).
A. Palop et al. / Int. J. Food Microbiology
29 (1996) I-10
9
According to our results, the influence of menstruum in the heat resistance of our strain is mainly due to pH (Table 3). The heat resistance in tomato and asparagus at pH 7 and 4 (Fig. 3A,B) was practically the same as that in McIlvaine buffer of these same pH’s. The effect of pH of food, as it happened with buffer, also decreased at higher temperatures of treatment and no statistically significant differences (p I 0.011 were detected either between .z values obtained with foods and with buffer at the same pH (Tables 2 and 4). However, in former investigations (Condon and Sala, 19921, it was observed that heat resistance of B. subtilis in food at its natural pH was somewhat different than that in buffer of the same pH. Maybe actual differences in behaviour among species or sporulation conditions could account for these differences. Remarkable influences of sporulation temperature on D, and z values of B. subtilis have been reported (Sala et al., 1995). No general conclusions can be drawn on the influence of pH of foods on z value, given the diversity of behaviours reported by investigators of the various species studied in different heating media. The acidification of heating menstruum, regardless of its composition, decreased heat resistance of our strain of B. Zicheniformis. But the magnitude of this decrease was smaller with increasing temperature being almost nil at the highest temperatures of treatment investigated.
Acknowledgements
This study was supported in part by Diputacion General de Aragon (Project P CA-5/89) which also provided a grant to A. Palop to carry out this investigation.
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Dawson, R.M.C., Elliot, D.C., Elliot, W.H. ano Jones, K.M. (1974) Data for Biochemical Research. Oxford at the Clarendon Press, Oxford, pp. 484-485. Fields, M.L., Zamora, A.F. and Bradsher, M. (1977) Microbiological analysis of home-canned tomatoes and green beans. J. Food Sci. 42, 931-934. Leitao, M.F.F., Ordorico, CA., Ciampi, C. and Quat, D.G. (1977) The thermal resistance of Bacillus stearothermophilus and Clostridium PA 3679 spores in banana puree. Cal. Inst. Technol. Aliment. 8. 313-327. Lowick, J.A.M. and Anema, P.J. (1972) Effect of pH on the heat resistance of Cl. sporogenes spores in minced meat. J. Appl. Bacterial. 35, 1199121. Mazokhina, N.N., Naidenova, L.P., Rozanova, L.1. and Dashevskaya, T.V. (1973) Heat resistance and pH effect on microorganisms, causing spoilage of canned foods. Acta Aliment. 2, 385-391. Mikolajcik, E.M. and Rajkowski, K.T. (1980) Simple technique to determine heat resistance of Bacillus stearothermophilus spores in fluid systems. J. Food Protect. 43. 799-804. Montville, T.J. and Sapers, G.M. (1981) Thermal resistance of spores from pH elevating strains of Bacillus licheniformis. J. Food Sci. 46, 1710-1712, 1715. Nakajo, M. and Moriyama, Y. (1993) Effect of pH and water activity on heat resistance of Bacillus coagulans spores. J. Jpn. Sot. Food Sci. Technol. 40, 268-271. Pflug, I.J. (19871 Factors important in determining the heat process value, F,, for low-acid canned foods. J. Food Protect. 50, 528-533. Pflug, I.J. and Odlaug, T.E. (1978) A review of Z and F values used to ensure the safety of low-acid canned foods. Food Technol. 32, 63-70. Rodriguez, J.H., Cousin, M.A. and Nelson, P.E. (1993) Thermal resistance and growth of Bacillus licheniformis and Bacillus subtilis in tomato juice. J. Food Protect. 56. 1655168. Sala, F.J., Ibarz, P., Palop, A., Raso, J. and Condon, S. (1995) Sporulation temperature and heat resistance of Bacillus subtilis at different pH’s. J. Food Protect. 58, 239-263. Steel, R.G. and Torrie, J.H. (19601 Principles and Procedures of Statistics. McGraw Hill Book Co. Inc., New York, pp. 161-180. Stumbo, CR. (1965) Thermobacteriology in Food Processing. Academic Press Inc., New York, pp. 98-102. Xezones, H. and Hutchings, I.J. (1965) Thermal resistance of Clostridium botulinum (62A) spores as affected by fundamental food constituents. Food Technol. 19, 113-l 15.