University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 8-2014 Casein Micelles and their Properties: Polydispersity, Association with Vitamin A and Effect of Ultra-High Pressure Homogenization Maneesha Sheenu Mohan University of Tennessee - Knoxville, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Dairy Science Commons, Food Chemistry Commons, and the Food Processing Commons Recommended Citation Mohan, Maneesha Sheenu, "Casein Micelles and their Properties: Polydispersity, Association with Vitamin A and Effect of Ultra-High Pressure Homogenization. " PhD diss., University of Tennessee, 2014. https://trace.tennessee.edu/utk_graddiss/2844 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Maneesha Sheenu Mohan entitled "Casein Micelles and their Properties: Polydispersity, Association with Vitamin A and Effect of Ultra-High Pressure Homogenization." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Food Science and Technology. Federico M. Harte, Major Professor We have read this dissertation and recommend its acceptance: Juan Luis Jurat-Fuentes, Qixin Zhong, Svetlana Zivanovic Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) Casein Micelles and their Properties: Polydispersity, Association with Vitamin A and Effect of Ultra-High Pressure Homogenization A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Maneesha Sheenu Mohan August 2014 DEDICATION Dedicated to my grandfather for his ‘Never say die’ attitude. ii ACKNOWLEDGEMENTS I am grateful to Dr. Federico Harte for providing me the opportunity to pursue my PhD at UT in a subject of great interest to me. I greatly appreciate my committee members Dr. Qixin Zhong, Dr. Svetlana Zivanovic and Dr. Juan Jurat-Fuentes, for their constant cooperation and guidance. I appreciate all the help provided by Dr. John Dunlap for performing cryo-TEM, TEM and STEM. I would also like to thank Dr. Michael Davidson for keeping me motivated for the college bowl competition even towards the end of my PhD. I am also grateful to my parents who have motivated me to dream sky high. My father is my role model in science, who has polished my capabilities with a very strict hand and gave valuable suggestions in my dissertation and my scientific quest so far. I am immensely thankful for my mother’s support in everything I have done. I also appreciate my brother’s unfailing faith in me. I would like to express my deepest devotion to my grandmother who is always praying for my wellbeing and success. It would have been very hard for me to get this far without my roommate Ujjal Jaggi’s support; she is my friend, sister and confidante. I would like to thank Rajesh Meena, a special friend for teaching me not to concentrate on the small pitfalls in life. I am grateful for all my friends among graduate students at the Food Science department, who have brightened my days with silly jokes. I will always remember the crazy discussions around the table in the graduate room. I would like to thank them for tolerating my ranting in the most stressful of times. I would never forget the time I spend playing badminton with Qiumin early every morning. I owe a lot to my lab mates, Ray Trejo, Vinay Mannam, Virginia Artegoitia, Manpreet Cheema, and Marta Corzo Martinez, who were always very supportive, in spite of my extremely long presentations during lab meetings. Many of these special people are iii my closest friends and have stood with me through the hardest times of my life. I would like to thank Oliver Janevski for teaching me so many of the valuable lessons in life. Maneesha S. Mohan iv ABSTRACT The internal structure of casein micelles is not fully understood. In the present work, we explore some of the basic questions about casein micelles in bovine milk including its size distribution, native binding properties and effect of ultra-high pressure homogenization (ultra-HPH) on technological properties of casein micelles. The size distribution of casein micelles was studied by cryo-TEM using skim milk from four cows. The degree of variation in casein micelle sizes (polydispersity) ranged between 0.39 and 0.47 for a size distribution ranging from 10 to 693 nm. These results indicated high polydispersity of casein micelles in milk from single cows. Casein micelles associated with vitamin A in four pasteurized skim milks (1.6 - 2.5 micro gm/mL of milk; 14 - 40% of the initial quantity detected in milks), while other protein fractions contained negligible vitamin A. Thus, casein micelles can inherently associate with hydrophobic probes like vitamin A in milk. This association of vitamin A to casein micelles in milk provided protection from degradation on exposure to ultraviolet light when compared to apple juice. Further, the effect of ultra-HPH up to 500 MPa on the physicochemical (apparent casein micelle size by dynamic light scattering) and technological characteristics (rennet coagulation - firmness [ 90 min, 1Hz, 0.01% strain]; acid gelation using 3%w/v glucono delta lactone – firmness [22°C, 110 min, 1Hz, 0.01% strain], SDS-PAGE) of casein micelles were studied. Casein micelle size increased from ca. 180 nm at 100 MPa to ca. 280 nm at 500 MPa HPH pressure. With increase in HPH pressure, renneting ability decreased until no coagulation was obtained for 500 MPa HPH milk. The firmness of HPH milk acid gels increased from about 76 Pa to 108 Pa when pressure was increased above 100 MPa (up to 400 MPa) HPH as compared to acid gels made from non-homogenized milk. Overall we elucidated the size distribution, binding ability to v vitamin A and changes occurring on ultra-HPH in casein micelles. This information can be utilized by the industry to modify and utilize casein micelles as an ingredient for different end uses. vi TABLE OF CONTENTS CHAPTER 1: Introduction and literature review ................................................................... 1 Introduction ................................................................................................................... 2 Casein micelles: structure and function ............................................................................. 2 Casein fractions .......................................................................................................... 2 Micelle size ................................................................................................................ 5 Structure of casein micelles ......................................................................................... 6 Functions of casein micelles ...................................................................................... 10 Rheological properties of casein micelles .................................................................... 12 Binding of casein micelles with hydrophobic probes ........................................................ 13 Caseins as nanocarriers ............................................................................................. 13 Modes of interaction facilitating delivery tasks ............................................................ 15 Hydrophobic probes for binding with caseins and casein micelles .................................. 16 Conditions affecting hydrophobic binding of casein micelles ........................................ 19 Effect of high pressure homogenization (HPH) on the physico-chemical properties of milk . 22 Pressure treatment studies .......................................................................................... 22 Principles of high pressure processing ......................................................................... 22 High pressure homogenization equipment ................................................................... 24 Effect of HPH on the properties of milk ...................................................................... 24 Effect of HPH on the physico-chemical properties of milk ............................................ 26 Effect of HPH on technological properties of milk. ...................................................... 34 Summary and objectives ............................................................................................... 40 References ................................................................................................................... 41 CHAPTER 2: Size distribution of casein micelles in raw skim milk from individual cows as studied using cryo-TEM ................................................................................................... 64 Abstract ...................................................................................................................... 65 Introduction ................................................................................................................. 66 Materials and methods .................................................................................................