Document Type

Thesis - Open Access

Award Date


Degree Name

Master of Science (MS)


Dairy Science

First Advisor

Sanjeev Anand


Biofilm, Italian Cheese, NSLAB, Pediococcus, Ripening, Sporulation


The first study was conducted on commercial Italian semi-hard cheese samples that were analyzed microbiologically to understand the effect of fast and slow rate of cooling on cheese microflora during ripening process at different temperatures. A cheese plant noticed an active growth of non-starter lactic acid bacteria, especially the heterofermentative bacteria, in ripened cheese blocks, which weren’t fully cooled. For that, three cheese sample sets, each having 8 samples, were received from the cheese plant for microbiological analysis. The first set included a group of fast cooled cheese samples to 38°F, and the other group was slow cooled to 50°F. The second set was cooled as the first set but further ripened for 2 months at either 38°F or 50°F, while the third set was ripened for 6 months after cooling. We were targeting the desired and undesired non-starter lactic acid bacteria (NSLAB), the total aerobic viable count for mesophiles / thermophiles, the thermoduric mesophiles / thermophiles, gas producers, Leuconostoc spp., Lactobacillus wasatchensis, and Clostridium spp. Non-selective and different types of selective media were used in order to enumerate and isolate each type of microorganism. The media were incubated aerobically or anaerobically at different temperatures depending on the targeted isolate’s optimum growth conditions. Random colonies on the selective media were picked and sent to another analytical laboratory for further analysis using Matrix Assisted Laser Desorption Ionization mass spectrometry - Time of flight technique MALDI-TOF. The total aerobic counts showed, in general, a declining trend during 6 months of ripening. Initial NSLAB counts in the 12 days old samples cooled to 38°F started at 5.87°0.13 logs CFU/g, and 6.19°0.13 logs CFU/g in the samples cooled to 50°F, and the two were significantly different. The fast-cooled samples thus showed lower counts than the slow cooled samples. According to MALDITOF identification, the added adjunct cultures were dominating in the whole study, which were Pediococcus acidilactici, Lactobacillus paracasei, and Lactobacillus rhamnosus. None of the samples had any of the Clostridium spp., undesired Lactobacillus spp. or Leuconostoc spp. Our results suggest that if accelerated ripening at 50°F is the goal, the rate of cooling to 38°F or 50°F will not make a difference from the bacterial count aspect in the presence of Pediococcus acidilactici as adjunct. However, bacterial distribution in cheese due to different rate of cooling might matter the most. The second study was aimed to understand the sporulation within biofilm matrix that formed on stainless steel surface by common sporeformers in the dairy industry. Undisturbed biofilm works as a reservoir for spore forming contaminants that might produce spores within the biofilm matrix itself. The results of this study showed that the duration of the biofilm formation and sporulation is variable among the different species and their strains. A strain of B. licheniformis ATCC⌐6634™ formed biofilm within 4 hours, but formed no spores even after an extended duration of incubation up to 21 days. However, the second strain of G. stearothermophilus ATCC⌐ 15952™ not only formed biofilms in 4 hours, but also sporulated within the biofilm matrix in 48 hours. On the other hand, B. licheniformis ATCC®14580™ took 7 days to produce spores within the NFDM and the biofilm. Keeping in mind the application of our research for future projects, the first study was conducted with cheese samples that contained high levels of Pediococcus acidilactici as a part of the adjunct cultures, which did not allow true predominance to emerge. It may be more useful to conduct this study without added Pediococcus, so that actual microbial interactions in cheese matrix could show up. The second study provided a proof of concept on the sporulation behavior of sporeformers within biofilm matrix. This would be useful to design cleaning strategies for resilient biofilms formed on plate heat exchangers and other equipment in dairy plants.


Includes bibliographical references



Number of Pages



South Dakota State University


In Copyright - Educational Use Permitted

Available for download on Friday, August 23, 2019

Included in

Microbiology Commons