Differentiation of lactate-fermenting, gas-producing Clostridium spp. isolated from milk

Endospores of Clostridium spp. capable of producing gas in a lactate-containing medium were enumerated from 14 pasteurized milk samples from Wisconsin cheese plants. Concentrations of endospores of lactate-fermenting, gas-producing Clostridium spp. were between 5.0×10−2 and 1.7×100 MPN ml−1. Concentrations of presumptive C. tyrobutyricum endospores (defined by subterminal endospore position and lactate dehydrogenase activity) were lower, not exceeding 2.0×10−2 MPN ml−1. Based on subterminal endospore position, lactate dehydrogenase activity, and a carbohydrate fermentation profile identical to C. tyrobutyricum strain ATCC 25755, five isolates (Ct) were initially characterized as C. tyrobutyricum, a known cause of late-blowing in high-pH cheeses. Twenty-eight other isolates (Cx) produced gas from lactate, but differed from ATCC 25755 in either endospore position, lactate dehydrogenase activity or carbohydrate fermentation profile. When inoculated at high concentrations in Gouda cheese, strain ATCC 25755, two Ct isolates and 18 Cx isolates tested produced gas during ripening. Among the five Ct isolates obtained and two reference strains confirmed as C. tyrobutyricum, there were four qualitatively different volatile organic acid byproduct profiles. Each of the two confirmed C. tyrobutyricum reference strains and five Ct isolates had distinct quantitative cell membrane fatty acid (CMFA) profiles. The Cx isolates represented 14 different volatile organic acid byproduct profiles and each isolate had a unique CMFA profile. Pulsed field gel electrophoresis (PFGE) of DNA from the two confirmed reference C. tyrobutyricum strains, four Ct and three Cx isolates, showed a low degree of relatedness. The results of this study suggest that a heterogeneous group of lactate-fermenting, gas-producing Clostridium spp. may be found in milk. Gas chromatographic analysis of volatile organic acid byproducts or CMFA, and PFGE of DNA are highly discriminating methods for differentiating Clostridium spp. that may cause late blowing in high-pH cheeses.