Toxin Production by Clostridium botulinum in Pasteurized Milk Treated with Carbon Dioxide

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The addition of carbon dioxide to milk at levels of <20mM inhibits the growth of selected spoilage organisms and extends refrigerated shelf life. Our objective was to determine if the addition of CO2 influenced the risk of botulism from milk. Carbon dioxide was added to pasteurized 2% fat milk at approximately 0, 9.1, or 18.2mM using a commercial gas-injection system. The milk was inoculated with a 10-strain mixture of proteolytic and nonproteolytic Clostridium botulinum spore strains to yield 101 to 102 spores/ml. Milk was stored at 6.1 or 21°C for 60 or 6 days, respectively, in sealed glass jars or high-density polyethylene plastic bottles. Milk stored at 21°C curdled and exhibited a yogurt-like odor at 2 days and was putrid at 4 days. Botulinal toxin was detected in 9.1mM CO2 milk at 4 days and in all treatments after 6 days of storage at 21°C. All toxic samples were grossly spoiled based on sensory evaluation at the time toxin was detected. Although botulinal toxin appeared earlier in milk treated with 9.1mM CO2 compared to both the 18.2mM and untreated milk, gross spoilage would act as a deterrent to consumption of toxic milk. No botulinal toxin was detected in any treatment stored at 6.1°C for 60 days. At 6.1°C, the standard plate counts (SPCs) were generally lower in the CO2-treated samples than in controls, with 18.2mM CO2 milk having the lowest SPC. These data indicate that the low-level addition of CO2 retards spoilage of pasteurized milk at refrigeration temperatures and does not increase the risk of botulism from treated milk stored at refrigeration or abuse temperatures.