Document Type

Dissertation - Open Access

Award Date


Degree Name

Doctor of Philosophy (PhD)


Dairy Science

First Advisor

Sanjeev Anand


Listeriosis is a life-threatening infection caused by foods contaminated with Listeria monocytogenes. Some of the major ice cream recalls in recent years reaffirm the ability of this food-borne pathogen to survive in diverse dairy processing environments and cause cross contamination. Inspection reports revealed certain lapses in implementing adequate hygienic practices for Listeria persistence in the processing environment, leading to cross contamination of ice cream. The higher levels of cross contamination of raw ice cream mix might result in random heat-injured cells when exposed to minimum heat treatment (69°C for 30 min). Evidence about the presence of injured cells in ice cream mix may thus prove useful to establish the overall Listeria risk, which was the aim of this study. Challenge studies were conducted to evaluate the dose dependent presence of heat-injured cells of Listeria. Ice cream mix formulations of 4 different types (36, 40, 42, and 45% total solids) were inoculated at 2.0, 3.0, and 4.0 log cfu/g levels of Listeria innocua (an established surrogate). The dose levels were selected based on a likely cross contamination on the raw side from environmental Listeria, especially due to their resident nature and growth in harborage sites. The samples were exposed to minimum heat treatment (69°C for 30 min) and the survivors, including heat-injured cells, were enumerated using standard protocols. A binary logistic regression model was fitted for evaluating the severity of risk. The influence of total solids, water activity, and pH variability were also studied on Listeria survival. The enrichment protocol, using buffered Listeria enrichment broth, followed by plating on modified oxford (MOX) agar and Rapid L’mono (RLM) medium, revealed the random presence of heat-injured cells in buffered Listeria enrichment broth, only at the highest dose level of 4+logs. Any potential risk from heat-injured cells was thus limited only to the highest levels of cross contamination, irrespective of the type of the mix. Significantly, none of the pasteurized ice cream mix samples supported the recovery of any heat-injured cells of Listeria during 72h holding at 7°C, even at the highest dose level of 4+logs, under the conditions of experimentation. Based on this part of the study, the level of cross contamination (dose) emerged as a predictor of the potential presence of heat-injured cells of Listeria when exposed to minimum pasteurization treatment. In the second phase of the study, we proposed a novel concept involving the possible protective role of air cells in the random presence of heat-injured cells. Challenge studies were conducted by inoculating ice cream mix samples (42% total solids) with L. innocua at a mean spiking level of log 4.0 cfu/g. The inoculated samples were subjected to minimum batch pasteurization treatment at 69°C for 30 min, and any heat-injured cells were enumerated using buffered Listeria enrichment broth (BLEB), followed by plating on MOX and RLM agar. Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) were conducted on the air-dried, spiked ice cream mix samples, at pre- and post-thermal treatment stages, which showed the presence of large air cells with some entrapped cells of Listeria. In the post heat- treated mix, SEM and AFM micrographs showed entrapped cells only within the larger air cells, whereas the mix matrix, did not show the presence of any Listeria cells. These observations thus suggested that the Listeria cells entrapped within the larger air cells might not have received adequate thermal effect and resulted in their random presence as heat-injured cells, as detected by enrichment protocol. In the third phase of the study we compared the effect of any temperature abuse of ice cream with the exposure to simulated gastro-intestinal fluids on the recovery potential of any carried over injured cells. Ice cream mix samples with injured cells were used in this study. Direct plating on Listeria selective agars was used for enumerating intact cells, while the heat-injured cells were recovered using BLEB, prior to enumerating on selective media. Although no intact cells were observed, the enrichment protocol revealed the random presence of heat-injured cells at the post-pasteurization stages of processing. Freezing and hardening steps did not appear to have any further detrimental impact on heat-injured cells, carried over from the pasteurized ice cream mix. The temperature abuse conditions, evaluated in the current study, although led to pudding consistency of ice cream, did not support the recovery of heat-injured cells. This thus implies that the post-pasteurization contamination with intact Listeria might pose a greater risk than any carried over injured cells. Similarly, such injured cells did not show any recovery in the simulated gastro-intestinal fluids tested under in vitro conditions. In the case of spiked intact cells, no detrimental effect of freezing and hardening steps was observed. Results from this study emphasize a need to design stage-specific critical control points to prevent any potential Listeria outbreaks.




South Dakota State University


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Available for download on Friday, April 15, 2022