Document Type

Dissertation - Open Access

Award Date


Degree Name

Doctor of Philosophy (PhD)

Department / School

Dairy Science

First Advisor

Sanjeev Anand


Bacillus, Drying, Fluorescence, Pilot study, SMP, Spores


This dissertation includes modeling of sporulation and germination behavior of Bacillus licheniformis strains during raw milk holding, tracking the survival of sporeformers and spores of high sporulating Bacillus strain during skim milk powder manufacturing. In addition, preliminary studies were done to standardize a rapid spore detection technique by using ratiometric fluorescence. To understand the role of strain variability during raw milk holding conditions, population dynamics of two strains of Bacillus licheniformis, ATCC 6634 and 14580, were modeled as a function of temperature (4.0 -12.0˚ C) and duration (0 - 72 h), using regression analysis. Based on initial spiking of vegetative cells (approx. 4.0 log cfu/mL) and spores (approx. 2.0 log cfu/mL), regression equations elucidating B. licheniformis growth behavior during raw milk holding at low temperatures were obtained. Contour plots were developed to determine the time-temperature combinations, keeping the population changes to less than 1.0 log. Results suggested that for vegetative cell spiking study of B. licheniformis ATCC 6634 (S1), cell population changes remained below 1.0 log up to 72 h at 8˚ C. For B. licheniformis ATCC 14580 (S2), 1.0 log shift was observed only after 80 h at 8˚ C, indicating greater multiplication potential of S1 as compared to S2. As S2 was a readily sporulating strain, the vegetative spiking study showed spore formation at different storage temperatures. In the presence of equivalent numbers of both types of sporulating strains in raw milk, despite strain variability, holding the milk at 8˚ C for not more than 72 h would keep any cell population changes below 1.0 log. In addition, under these storage conditions, the population would remain as vegetative cells that could easily be inactivated by pasteurization. As BL ATCC 14580 was the high sporulating strain, therefore, further studies were conducted using this strain, wherein spiked milk samples were held prior to manufacturing of milk powder. A pilot-scale skim milk powder trial was conducted to evaluate the influence of holding conditions on final spore and sporeformer counts. After spiking, raw milk silos were maintained at two different holding temperatures i.e. at PMO based conditions (10˚ C for 4 h, followed by 7˚ C for 72 h, treatment 1) and at optimum holding condition based on contour plots (4˚ C for 24 h, treatment 2). Powders manufactured under these conditions were assessed for vegetative and spore population at different stages of processing. The overall final spore and vegetative cell counts in the powders manufactured under optimum holding condition (4˚ C for 24 h) were found to be significantly lower (0.58 ± 0.04 and 1.82 ± 0.05 log cfu/g) as compared to the conditions likely to practice by dairy plants (2.74 ± 0.03 and 1.03 ± 0.06 log cfu/g). This shows that milk powders with reduced vegetative and spore counts can be prepared by optimizing the raw milk holding conditions. For standardization of a novel spore detection method, CaDPA content of spores was quantified using a ratiometric fluorescence technique. This method is based on the detection of CaDPA that enhances the luminescence of lanthanide ion when complexed with a semiconducting polymer. The intensity was recorded after chelating semiconducting fluorescent polyfluorene (PFO) dots with terbium ions, sensitized by different volumes of CaDPA (0.1μM). The standard curve showed a linear relationship (R2 = 0.98) in the experimental concentration range of 2.5 nM to 25 nM of CaDPA, with corresponding intensity (a.u.) of 545 to 2130 nm. In HPLC grade water, the minimum log spores detected were 1.36 ± 0.09 log cfu/mL with corresponding mean CaDPA content ofhand, for raw skim samples, the minimum log spores detected were 5.21 ± 0.07 cfu/mL with mean CaDPA content of approx. 2.0. For raw milk spiked samples, reduced fluorescence detection was observed and was approx. five times lower as compared to the spiked samples of HPLC grade water. The reduced fluorescence ability in raw milk can be due to the turbidity of the solution or interference of proteins, amino acids and other ions of milk. This study provides a proof of concept for a potential application of this technique to rapidly detect bacterial endospores in the dairy and food industry. Further studies are required to remove the inference of ionic components in milk to improve the efficiency of the protocol. Based on these studies we were able to establish the holding time-temperature values for raw milk holding that would result in the least change in the population of sporeformers and spores. Using these combinations, we were able to manufacture skim milk powder with lower counts of spores and sporeformers as compared to the PMO recommended holding conditions. The proof of concept generated through the spore detection protocol has the potential to be established as a rapid detection technique for spore counts in milk and related products.

Library of Congress Subject Headings

Milk -- Microbiology.
Dried skim milk.
Bacillus (Bacteria)
Sporeforming bacteria.
Dairy processing -- Quality control.



Number of Pages



South Dakota State University



Rights Statement

In Copyright