Thesis--Rijksuniversiteit te Utrecht. / Summary in Dutch. Includes bibliographical references (p. 87-98).

Shelf-stable canned butter is currently available in retail stores, and many home-preservationists promote home-canning of butter. Non-cultured butter is a low-acid canned food, which would presumably require thermal processing. The lack of a thermal process step in canned butter products raises questions of potential safety, because they are hermetically sealed and generally exhibit anaerobic growth conditions, which are optimal for Clostridium botulinum growth. Without thermal processing, low-acid canned foods (LACF) must have inhibitory factors present to prevent C. botulinum growth. Some potential intrinsic inhibitory factors, or "hurdles", within butter include: reduced water activity (aw), acidity (pH) in cultured products, elevated salt content, and the micro-droplet nature of the aqueous phase in the butter emulsion. It was hypothesized that a normal intact butter emulsion would have sufficient "hurdles" to prevent C. botulinum growth, while a broken butter emulsion would result in a larger aqueous phase that would allow for growth. Butter was prepared using a batch churn method with either inoculated or uninoculated cream. Butter samples with four different salt amounts (0, 0.8, 1.6, & 2.4% added NaCl) were prepared and placed in coated aluminum cans for storage. Samples were stored for 1 or 2 week periods at either 22°C or 41°C and then plated for C. sporogenes growth. Samples stored at 41°C showed a significant increase over those stored at 22°C. This growth increase occurred due to incubation near the optimal growth temperature for C. sporogenes and damage to emulsion structure. Furthermore, sodium chloride (NaCl) addition was found to have a significant effect on C. sporogenes growth, with 0.8 % NaCl promoting more growth than 0%, but with decreases in growth beyond 0.8%. Uninoculated control plates were also found to have bacterial growth. This growth was attributed to other anaerobic bacteria present within the cream. It was concluded that removal of the butter structure "hurdle" could result in C. botulinum growth even at elevated salt levels and therefore home preparation of canned butter is not advisable. It is also possible that commercially canned butter, if heat abused, could potentially allow for C. botulinum growth and therefore consumption is not recommended.

A procedure to rapidly screen Clostridium botulinum type A and Clostridium sporogenes for plasmids was developed. Plasmid profiles of five C. botulinum type A strains and seven C. sporogenes strains were determined and a possible relationship of plasmids to toxin production was examined. The differentiation of these organisms by plasmid fingerprinting was also studied. The plasmid isolation procedure was a modified cleared lysate technique, including lysis under alkaline conditions. Samples were subject to agarose gel electrophoresis to detect plasmid DNA. Culture age affected plasmid detection due to changes in the cell density and lysing efficiency. Middle to late log cultures were used throughout the study because they provided optimum plasmid detection. Four out of five C. botulinum type A strains and three out of seven C. sporogenes strains contained extrachromosomal DNA. For those C. botulinum type A strains which contained plasmids, there were always two, one 15 to 15.6 Mdal and the other 6.2 Mdal. C. sporogenes showed less consistency in plasmid size and number and their plasmids were generally of a greater molecular weight than those in C. botulinum type A. One C. sporogenes strain contained two plasmids and two strains contained one plasmid. Toxin production may be plasmid-mediated in the plasmid containing strains, but there was no apparent general relationship, because one of the toxic strains did not show the presence of plasmids. Plasmid screening may be useful in the differentiation of these closely related organisms without toxin testing. / Master of Science

Clostridium sporogenes PA 3679 spores were inoculated into a meat-vegetable mixture before extrusion, cooking, and vacuum packaging into "stewsticks" to simulate Clostridium botulinum growth. The experiment was a 3 x 5 x 2 x 3 factorial which determined the influence of pH, water activity, initial spore load, and storage period on spore survival. Spore levels decreased throughout storage for all treatments. Spore levels decreased linearly (P = 0.02) as water activity increased, in samples that were heated to kill vegetative cells and activate spores. Other significant interactions of heat-treated samples were observed with inoculum level (P < 0.01) and storage time (P < 0.01). Spore levels in stored products were also significantly affected by water activity* inoculum level (P = 0.03), pH * time, water activity* time (P = 0.01), inoculum level * time (P < 0.01), and water activity * inoculum levels * time (P < 0.01). The interaction between pH * water activity * time tended towards significance (P = 0.06). Most probable number estimates in nonheated samples accounted for naturally occurring viable cells and spores, and added spores and were significantly affected by the main effects of inoculum level (P < 0.01) and time (P < 0.01). The two-way interactions of water activity * inoculum level (P = 0.04), pH * inoculum level (P < 0.01),water activity * time (P < 0.01), and three-way interaction of pH * inoculum level * time (P = 0.03) were significant. Spore levels approached 102, or less (compared to an inoculum level of 106 spores per gram) due to the effects of many treatments. Some stewstick packages were observed to become "gassy" or "loose" during storage. Subsequently the stewstick packages were used to isolate microorganisms that were able to grow at water activities of 0.96-0.86, in glycerol-adjusted Rogosa agar, and were acid tolerant to pH 4.4-4.2. One produced gas in pure culture, and some produced indole. These bacteria were not destroyed by heating to 74°C for at least 30 minutes, and lowered the pH in the stewstick during storage. In conclusion, in all stewstick samples, regardless of pH or Aw, inoculated clostridial spore levels decreased during storage, apparently because spores germinated and vegetative cells subsequently died. Thus, if stewsticks are cooked to 74°C throughout, have a Aw ≤ 0.86 and pH ≤ 4.8, they appear to be safe.