Clostridium botulinum growth and toxin production in tomato juice containing Aspergillus gracilis

Abstract

The ability of spores of one type A and one type B strain of Clostridium botulinum to grow and produce toxin in tomato juice was investigated. The type A strain grew at pH 4.9, but not at pH 4.8; the type B strain grew at pH 5.1, but not at pH 5.0. Aspergillus gracilis was inoculated along with C. botulinum spores into pH 4.2 tomato juice; in a nonhermetic unit, a pH gradient developed under the mycelial mat, resulting in C. botulinum growth and toxin production. In a hermetic unit, mold growth was reduced, and no pH gradient was detected; however, C. botulinum growth and low levels of toxin production (<10 50% lethal doses per ml) still occurred and were associated with the mycelial mat. The results of tests to find filterable or dialyzable growth factors were negative. It was demonstrated that for toxin production C. botulinum and the mold had to occupy the same environment. chosphoron sp., and Cladosporium sp. had grown and shifted the pH from <4.6 to >4.6. These are typical cases of metabiosis. This study was carried out to deternine whether C. botulinum growth and toxin production would occur in the presence of Aspergillus sp. in a specific microenvironment within a food system where the pH was less than 4.6. (The results are from a Ph.D. thesis submitted by Theron E. Odlaug to the faculty of the Graduate School of the University of Minnesota.) MATERLS AND METHODS C botulinum spores. Cultures of a C. botulinum type A strain, A16037, and a type B strain, B15580, implicated in outbreaks of botulism where homecanned tomato products were the toxin-carrying vehicle (3, 4), were obtained from the Center for Disease Control in Atlanta, Ga. Spore crops were prepared from these strains by using methods described by Odlaug and Pflug (13). Aspergillus gracilis spores. The strain of mold used in these studies was isolated from a l-quart (ca. 0.95-liter) jar of spoiled stewed tomatoes. The jar was obtained from I. D. Wolf (Department of Food Science and Nutrition, University of Minnesota). The mold was identified as A. gracilis by morphological characteristics, using culture techniques described by Thom and Raper (18). Conidiospore crops were prepared by using potato dextrose agar (Difco). The procedure was the same as that used by Buchanan et al. (1) for growing conidiospores ofAspergillus flavus. A 300-nil flask containing 75 ml of potato dextrose agar was inoculated from the A. gracilis stock culture and incubated at 22°C for 21 days. After incubation the spores were harvested by add-496

Metabiotic Association of Fusarium, Alternaria, and Rhizoctonia with Clostridium botulinum in Fresh Tomatoes

“…This effect was also seen with Rhizoctonia at day four, but no gradient in pH was found with Altemaria. Similar gradients have been observed in inoculated, sterilized tomato juice (Huhtanen et al, 1976;Odlaug and Pflug, 1979).…”

“…These observations lead to speculation that a metabiotic association may occur in fresh tomatoes involving molds and C. botulinum. Such a relationship has been observed in test tube studies with heated juice (Hutanen et al, 1976;Odlaug and Pflug, 1979). Therefore, this study was undertaken to determine whether growth of fungi in fresh tomatoes could create a situation in which C. botulinurn or C. sporogenes could grow in the fresh fruit and to determine if C. botulinurn could produce toxin in a metabiotic relationship with the fungi.…”

“…However, not all strains of the bacterium will germinate at this low pH. Odlaug and Pflug (1979) and Dozier (1924) observed strains not capable of growth below pH 5.0. Strain 62A of the National Canners Association also was incapable of growth in tomatoes below pH 5.0 (NCA, 1965).…”

Growth and toxin formation by Clostridium botulinum at low pH values

“…In a detailed study of the growth of Cl. botulinum in tomato juice, Odlaug & Pflug (1979) found that type A could grow and form toxin at pH 4.2 in tomato juice if Aspergillus gracilis was inoculated together with Cl. botulinurn.…”

Metabiosis of proteolytic moulds and Salmonella in raw, ripe tomatoes

“…The pH of commercially processed tomato juice increased by an average of 2AE3 units (mean pH 6AE4) from an initial pH of 4AE1 when juice was inoculated with proteolytic fungi. Other researchers have observed that fungi commonly involved in spoilage of tomatoes are capable of raising the pH of tomato juice and tissue to levels that enable Clostridium botulinum to grow (Huhtanen et al 1976;Mundt 1978;Odlaug and Pflug 1979;Draughon et al 1988).…”