Nov 22, 1982 - media with a water activity (aw) of 0.972 or 0.965 and a pH of5.7, but no growth or .... bic agar (Difco Laboratories, Detroit, Mich.) medium (with ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1983, p. 1150-1152
Vol. 45, No. 3
0099-2240/83/031150-03$02.00/0 Copyright © 1983, American Society for Microbiology
Clostridium botulinum Type A Growth and Toxin Production in Media and Process Cheese Spread JORGE BRIOZZO,1 ETHEL AMATO DE LAGARDE,2 JORGE CHIRIFE,3* AND JOSE L. PARADA3 Comisi6n de Investigaciones Cientificas de la Provincia de Buenos Aires,1 Instituto Nacional de Microbiologia "Dr. Carlos G. Malbrdn, "2 and Departamento de Industrias and Departamento de Quimica Biol6gica, Facultad de Ciencias Exactas y Naturales, Ciudad Universitaria, 1428 Buenos Aires,3 Argentina
Received 21 September 1982/Accepted 22 November 1982
We found that Clostridium botulinum type A grew well and produced toxin in media with a water activity (aw) of 0.972 or 0.965 and a pH of 5.7, but no growth or toxin production was observed at or below an a, of 0.949 during incubation at 30°C for 52 to 59 days. aw and pH values of media were adjusted to those of cheese spreads commercially produced. Solutes used to adjust aw included combinations of NaCl, cheese whey powder, emulsifying salt, sodium tripolyphosphate, and glycerol. In agreement with results obtained for media, toxin was produced in samples of cheese spread (aw, 0.970; pH, 5.7) at 30 to 70 days of incubation at 300C.
Early in 1974, Argentine authorities reported an outbreak of Clostridium botulinum type A botulism in Buenos Aires which was caused by a commercial cheese spread with onions and which involved six cases and three deaths (3). A survey of the water activity (a,) and pH values of Argentine commercial cheese spread has been made (R. C. Magrini, J. Chirife, and J. L. Parada, J. Food Sci., in press). The a, values of cheese spreads varied between 0.968 and 0.978, and the pH values ranged from 5.6 to 6.1. On the basis of a, and pH, it seems that if other factors are favorable, all of these cheeses are theoretically capable of supporting C. botulinum growth and toxin production. The aim of this study was to determine whether C. botulinum type A spores could germinate, grow, and produce toxin in nonrefrigerated process cheese spread. For this purpose, we first studied growth and toxin production in synthetic media with a, and pH values adjusted to those of cheese spreads produced in Argentina. Different solutes, alone or in combination, were used to adjust a,, since it is known that the initiation of growth by C. botulinum spores depends not only on the a, of the medium but also on the nature of the solute used. Solutes such as NaCl, cheese whey powder, and emulsifying salts (phosphate based) were used to depress a, since they may be included in commercial formulations of cheese spreads (6). The results were confirmed by studies of toxin production in an inoculated commercially prepared cheese spread. C. botulinum type A strain INM 9 (obtained from the collection of the Instituto Nacional de Microbiologia "Dr. Carlos G. Malbran," Buenos
Aires) was used in all experiments. The basal (modified TPG) medium used for all experiments contained 25 g of Trypticase (BBL Microbiology Systems), 5 g of yeast extract, 5 g of glucose, 0.5 g of cysteine, and 1,000 ml of distilled water (final pH, 7.3; aw, 0.996). The media were adjusted to the required a, values by the addition of one or more of the following compounds: NaCl, glycerol, cheese whey powder (protein, 11 to 12%; lactose, 73 to 76%; fat, 1%; moisture, 4 to 5%), sodium tripolyphosphate (STP), and Joha S-9 emulsifying salt (Hoeschst; Buenos Aires, Argentina). The pH values of the media were brought to around 5.7 with HCl. Medium a, values were determined with an electronic hygrometer (model HM 14; Humicap, Vaisala, Finland) (G. Favetto, S. Resnik, J. Chirife, and C. Ferro Fontan, J. Food Sci., in press). Table 1 shows the compositions and a, and pH values of the media. The media were placed in screwcapped test tubes. We inoculated 0.2 ml of a heat-shocked spore suspension into each tube to obtain about 5 x 103 spores per ml. Anaerobiosis was secured by placing a layer of sterile paraffin on the surface of each medium. The cheese spread (a,, 0.970; pH, 5.7; moisture content, 56.0%; fat, 19%; protein, 11%; NaCl, 1.5%) was inoculated by thorough mixing with heat-shocked spores. Mixing was done in an Omni-Mixer (Ivan Sorvall, Inc., Norwalk, Conn.) under a stream of sterile nitrogen. The inoculum consisted of 102 or 104 spores per g of cheese. The media and the cheese spread samples were incubated at 30°C. Toxicity tests of each cheese spread sample were made with extracts prepared from a homogenized portion
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VOL. 45, 1983
NOTES
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TABLE 1. Descriptions of media'
Description of medium:
Ingredient/value234 3 4 2 1 Cheese whey powder (g) NaCl (g) Joha S9 emulsifying salt (g) STP (g) Glycerol (g)
14.75 1.97
9.66 1.52
9.66 1.52 5.08
5
14.46 2.37 8.97
14.43 4.02 8.97
1.97
1.97
5.08
0.965 0.938 0.972 0.965 0.949 5.7 5.7 5.7 5.8 5.6 a aw and pH values were adjusted to those of process cheese spreads. Each medium contained 100 g of basal medium.
aw,
pH
and extracted with an equal volume of sterile physiological saline solution. Two mice were inoculated intraperitoneally with 0.5-ml portions of appropriate dilutions of extracts from each sample. The procedure employed for toxicity tests with the various media was essentially the same. Spore and bacterial suspensions of C. botulinum were properly diluted in a sterile solution containing 1 g of tryptone, 8.5 g of NaCl, and 0.5 g of cysteine per liter. One-milliliter portions of the dilutions were inoculated into duplicate screw-capped tubes with melted Brewer anaerobic agar (Difco Laboratories, Detroit, Mich.) medium (with redox indicator) at a temperature of 42 to 45°C. The tubes were incubated at 30°C for 48 h, and the visible colonies were recorded. The inoculated cheese samples were removed from incubation at different times and thoroughly mixed with saline-tryptone-cysteine solution
in
an
Omni-Mixer under
a
sterile nitrogen
stream. A procedure similar to that described was then spores or viable
above
followed, and the numbers of cells per gram of cheese were
determined. Published data are not available on the a, values of Joha S-9 emulsifying salt and cheese whey powder. For this reason, we measured (2, 4) the a, values of Joha S-9 salt, cheese whey solutions, and STP (data not shown). Table 2 shows the behavior of C. botulinum type A in media with a, and pH values adjusted to those of process cheese spreads (media 1, 2, and 3) and in media with lowered a, values (media 4 and 5) incubated at 30°C. C. botulinum grew well and produced toxin in media with an a, of 0.972 or 0.965, although in the latter case, growth and toxin production were delayed, compared with growth and production in medium with an a, of 0.972. It is noteworthy that neither
TABLE 2. C. botulinum type A growth and toxin production in media incubated at 30°C Growth and toxin production in mediuma: Incubation Control' 5 4 3 2 1
time (days)
0 3 7 10 14 17 21 24 28 31 38 45 52
CFU/ml Toxinc CFU/ml Toxin 4.0 x 103 - 4.8 x 103 6.3 x 107 2,000 2.2 x 108 200 5.0 x 107 20,000 3.9 x 107 2,000 1.1 x 108 40,000 6.4 x 106 20,000 2.6 x 106 ND 2.2 x 106 ND
CFU/ml Toxin CFU/ml Toxin CFU/ml Toxin CFU/ml Toxin
5.2 5.6 1.0 2.2 2.0
x 103 - 4.7 x 103 - 5.4 x 104 ND 7.0 x 106 ND 1.3 20 1.9 x 106
x 103 x103
103 x 106 x 105
x
5.5 x 106 2,000 3.2 x 106
1.0 x 104 2,000 1.0 x 104 5.0 x 10 ND 1.2 x 105 6.0 x 104
59
Media are described in Table 1. b Basal medium (see text). c Minimal lethal dose per milliliter.-, None detected; ND,
a
not done.
9.0X 103 1.6x 104 ND 9.0 x 103 ND ND 1.6 x 104 ND >20 5.0 x 103 ND 1,000 9.0 x 103 ND 1,000 1.1 X 104 ND 1.6 x 104 ND ND 1.7 x 104 ND 1.2 x 104 ND 1.5 x 104 ND 7.0 x 103 ND 1.9 x 104-
3.5
-
6.2 x 1.0 x 6.2 x 8.0 x 1.7 x 6.5 x 5.0 x 8.8 x 9.0 x 6.0 x 9.0 x 1.4 x
x
103 103
-
104 ND 103 103 104 103
ND ND ND
ND
103 ND
103 103 103 103 103 1.2 x 104
-
ND ND ND ND
1152
NOTES
APPL. ENVIRON. MICROBIOL.
emulsifying salt nor STP prevented C. botuli- abuse, i.e., room temperature in tropical areas num type A growth or toxin production. When or during summer. This potential health hazard the a, was further depressed to 0.949 or 0.938 may be controlled by reducing the normal a, of (media 4 and 5), no growth or toxin production cheese spreads (ca. 0.97) by the addition of was observed, even after 52 to 59 days of adequate solutes. However, the feasibility and incubation at 30°C. On the basis of the results effectiveness of this procedure should be conobtained for media, one could expect that C. firmed by inoculated-pack studies of the final botulinum type A grows and produces toxin in formulated product. process cheese spread. Two samples of cheese We acknowledge financial support from Comisi6n de Invesspread (aw, 0.970; pH, 5.7) were inoculated with tigaciones Cientificas de la Provincia de Buenos Aires (Curso 102 or 104 spores per g and incubated at 30°C. de Ciencia y Tecnologia de Alimentos). We acknowledge the Instituto Nacional de Microbiologfa Toxin was produced in both samples at 30 to 70 days of incubation. Inoculum size did not affect "Dr. Carlos G. Malbran" for the use of its facilities. growth or toxin production; by the end of the LITERATURE CITED incubation period, both high- and low-inoculum 1. Baird-Parker, A. C., and B. Freame. 1967. Combined effect of water activity, pH and temperature on the growth of samples showed similar counts (5.0 x 104 to 9.0 Clostridium botulinum from spore and vegetative cell inx 104 CFU/g). Baird-Parker and Freame (1) ocula. J. Appl. Bacteriol. 30:420-429. minimal for growth reported that the a, required 2. Chirife, J., C. Ferro Fontan, and S. Vigo. 1981. A study of of C. botulinum type A when NaCl is used to water activity prediction for molasses solutions. J. Agric. Food Chem. 29:1085-1086. adjust a, is 0.96 at pH 7.0 and 0.97 at pH 5.5. Lagarde, E. A. 1974. Boletfn informativo del Centro Minimal a, values necessary for growth were 3. de Panamericano de Zoonosis, vol. 1. Centro Panamericano reported to be lower when glycerol was used de Zoonosis, Buenos Aires, Argentina. instead of NaCI (0.93 and 0.95 at pH values of 4. Ferro FontAn, C., and J. Chhrife. 1981. The evaluation of water activity in aqueous solutions from freezing point 7.0 and 5.5, respectively). Ohye and Christian J. Food Technol. 16:21-30. (5) found that in synthetic media, the minimal a, 5. depression. Ohye, D. F., and J. H. B. Christian. 1966. Combined effor growth of C. botulinum type A is 0.95 when fects of temperature, pH and water activity on growth and NaCl is used to depress a,. Our results for toxin production by Clostridium botulinum types A, B and E, p. 217. In Proceedings of the Fifth International Sympomedia and cheese agreed with these findings. sium on Food Microbiology, Moscow. It may be concluded that process cheese N., J. M. Gowpfert, E. Traisman, and W. M. spreads with the same a, and pH values used in 6. Tanaka, Hoffbeck. 1979. A challenge of pasteurized process cheese the present study support C. botulinum type A spread with Clostridium botulinum spores. J. Food Prot. 42:787-789. growth and toxin production under temperature
