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Som N. Khanal

Document Type

Thesis - University Access Only

Award Date


Degree Name

Master of Science (MS)

Department / School

Dairy Science

First Advisor

Sanjeev K. Anand


Thermoduric sporeformers and spores survive thermal treatment of milk and cause spoilages of dairy produces. Several non-thermal, thermal, or combined techniques have been studied to inactivate bacterial spores, but none of them are completely effective. Low frequency ultrasonication, has been known to produce intense cavitation that inactivates bacterial cells by disintegrating them. In the present investigation, ultrasonication was evaluated as a nonthermal processing technique to inactivate sporeformers and spores in milk. Vegetative cells of Bacillus coagulans (ATCC® 12245), Anoxybacillus flavithermus (DSM 2641 ), B. sporothermodurans (DSM 10599), B. licheniformis (ATCC® 6634), and Geobacillus stearothermophilus (ATCC® 15952) were studied for their survivability during batch pasteurization (63°C for 30 min) of spiked skim and whole milk samples. Experiments were also conducted to study the role of ultrasonication in induction of spore germination. Vegetative cells of Bacillus coagulans (ATCC® 12245), and Anoxybacillus flavithermus (DSM 2641) were found to survive pasteurization treatment in both skim and whole milk samples. On the other hand, B. licheniformis, B. sporothermodurans, and G. stearothermophilus were inactivated by pasteurization. Sporulation studies revealed that Bacillus coagulans (ATCC® 12245), B. licheniformis (A TCC® 6634), and Geobacillus stearothermophilus (ATCC® 15952) produced spores during incubation in agar plated for 1 to 2 weeks. On the other hand, Bacillus sporothermodurans (DSM 10599) and B. coagulans (ATCC® 7050) did not sporulate under the laboratory conditions. Ultrasonication at 80% amplitude for 10 minutes was observed as the most effective treatment against the thermoduric vegetative cells, as well as, spores. This treatment inactivated the vegetative cells of B. coagulans, A.jlavithermus and B. sporothermodurans in skim milk by 4.53 , 4.26, and 3.61 log cycles, respectively. Process of pasteurization (63°C/30min) after ultrasonication completely eliminated about log 6 cfu /mL (99.9999%) of these cells in skim milk. Similar ultrasonication treatment resulted in a maximum of 35.63, 33.23, and 48.96 % inactivation of the spores of B. coagulans, B. licheniformis and G. stearothermophilus, respectively. It was possible to enhance the spore inactivation to 50.39%, 37.88%, and 65.74 %, respectively, by combining pasteurization with ultrasonication. Combination of ultrasonication and higher heat treatment (80°C for 1 min) further enhanced the inactivation of G. stearothermophilus spores by 75. 32%. Significantly higher (P < 0.05) percentage of spores was inactivated by the combined treatments. Ultrasonication also showed induction of spore germination as evident by higher thermal inactivation of ultrasonicated spores after incubation. As visualized under the scanning electron microscope, vegetative cells of sporeformers were physically disintegrated after ultrasonication. On the other hand, virtually no changes were observed in the morphology of spores after either ultrasonication or combined ultrasonication and thermal treatments. Ultrasonication at 80% amplitude for IO minutes caused significant reduction (P<0.05) in brightness and greenness of milk; whereas, blueness (b*) of milk was increased. Similar ultrasonication treatment did not cause any change in the pH and alkaline phosphatase activity (P>0.05) of skim milk.

Library of Congress Subject Headings

Milk -- Microbiology
Milk contamination
Ultrasonic waves -- Industrial applications
Bacterial spores


Includes bibliographical references (pages 120-147)



Number of Pages



South Dakota State University


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