Casein Proteins as a Vehicle to Deliver Vitamin D3: Fortification of Dairy Products with vitamin D3 and Bioavailability of Vitamin D3 from Fortified Mozzarella Cheese Baked with Pizza by Banaz Al-khalidi A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Nutritional Sciences University of Toronto © Copyright by Banaz Al-khalidi (2012) Casein proteins as a vehicle to deliver vitamin D3: Fortification of dairy products with vitamin D3 and Bioavailability of vitamin D3 from fortified Mozzarella cheese baked with pizza Banaz Al-khalidi Master of Science Department of Nutritional Sciences, Faculty of Medicine University of Toronto 2012 ABSTRACT Current vitamin D intakes in Canada are inadequate. The extension of vitamin D fortification to additional foods may be an effective and appropriate strategy for increasing vitamin D intakes in the general population. Cheese is potentially an ideal candidate for vitamin D fortification. We introduce the potential use of casein proteins as a vehicle for vitamin D3 fortification in industrially made cheeses where we found that over 90% of vitamin D3 added to milk was retained in both Cheddar and Mozzarella cheeses. Use of casein proteins for vitamin D3 fortification did not fully prevent vitamin D3 loss into whey. However the loss was minimized to approximately 8%. We then show that vitamin D3 is bioavailable from fortified Mozzarella cheese baked with pizza suggesting that the high temperature baking process does not significantly breakdown vitamin D3. Our findings could have important implications in increasing fortified food options for Canadians. ii TABLE OF CONTENTS ABSTRACT ................................................................................................................................... ii LIST OF TABLES ........................................................................................................................ v LIST OF FIGURES ..................................................................................................................... vi CONTRIBUTIONS..................................................................................................................... vii ACKNOWLEDGEMENTS ...................................................................................................... viii CHAPTER 1 : INTRODUCTION ............................................................................................... 1 CHAPTER 2 : LITERATURE REVIEW .................................................................................. 5 2.0 VITAMIN D AND HEALTH ............................................................................................. 6 2.1 Historical Perspective ........................................................................................................ 6 2.2 Photobiology of Vitamin D ............................................................................................... 7 2.3 Vitamin D Absorption and Transport into Circulation ...................................................... 9 2.5 Physiologic Actions of Vitamin D ................................................................................... 12 2.6 Dietary Guidelines for Adequacy and Current Intakes ................................................... 13 2.7 Vitamin D Inadequacy in Canada .................................................................................... 16 2.8 Toxicity of Vitamin D ..................................................................................................... 17 2.9 FOOD FORTIFICATION ................................................................................................ 21 2.10 Food Fortification .......................................................................................................... 21 2.11 Advantages and Limitations of Food Fortification ........................................................ 22 2.12 Vitamin D Fortification of Food .................................................................................... 23 2.13 Rationale for Increased Vitamin D fortification ............................................................ 24 2.14 Rationale for Vitamin D3 Fortification of Cheese ......................................................... 26 2.15 Rationale for Binding of Vitamin D3 to Caseins ........................................................... 28 CHAPTER 3 : HYPOTHESES AND OBJECTIVES.............................................................. 30 3.0 Hypotheses and Objectives ............................................................................................... 31 3.1 Hypotheses....................................................................................................................... 31 3.2 Objectives ........................................................................................................................ 31 CHAPTER 4 : VITAMIN D3 FORTIFICATION OF INDUSTRIALLY MADE CHEDDAR AND MOZZARELLA CHEESES USING CASEINS AS A DELIVERY SYSTEM ........... 33 iii 4.0 Abstract ............................................................................................................................... 34 4.1 Introduction ......................................................................................................................... 35 4.2 Materials and Methods ........................................................................................................ 36 4.3 Results and Discussion ........................................................................................................ 42 4.4 Conclusion ........................................................................................................................... 48 CHAPTER 5 : THE BIOAVAILABILITY OF VITAMIN D3 FORTIFIED MOZZARELLA CHEESE UNDER HIGH TEMPERATURE PIZZA BAKING PROCESS ....................................................................................................................................................... 49 5.0 Abstract ............................................................................................................................... 50 5.1 Introduction ......................................................................................................................... 51 5.2 Materials and Methods ........................................................................................................ 52 5.3 Results ................................................................................................................................. 57 5.4 Discussion ........................................................................................................................... 64 CHAPTER 6 : GENERAL DISCUSSION ............................................................................... 68 REFERENCES ............................................................................................................................ 75 iv LIST OF TABLES Table 2-1 Vitamin D Dietary Reference Intakes (DRIs) for Adequacy (amount/day) ................. 15 Table 2-2 Tolerable Upper Intake Level of Vitamin D ................................................................ 19 Table 4-1 Chemical Composition of Milks and Wheys................................................................ 42 Table 4-2 Yields, Recoveries, and Chemical Composition of Fortified Cheeses ......................... 43 Table 4-3 Vitamin D3 Recovery in High Dose (28000 IU/serving) Cheddar cheese ................... 44 Table 4-4 Vitamin D3 Recovery in High Dose (28000 IU/serving) Mozzarella cheese ............... 44 Table 4-5 Stability of Vitamin D3 in Casein Premix through Storage at -20ºC............................ 47 Table 4-6 Stability and Distribution of Vitamin D3 in Mozzarella Cheese through Storage at - 20ºC ............................................................................................................................................... 48 Table 5-1 Demographics and baseline characteristics of subjects consuming vitamin D3-fortified Mozzarella cheese baked with pizza ............................................................................................. 58 Table 5-2 Biochemical responses in subjects consuming vitamin D3-fortified Mozzarella cheese baked with pizza ........................................................................................................................... 59 v LIST OF FIGURES Figure 2-1 The chemical structures of vitamin D2 and vitamin D3................................................. 8 Figure 2-2 Schematic representation of the synthesis and metabolism of vitamin D .................. 11 Figure 5-1 Trial profile of subjects enrolled in the study ............................................................. 54 Figure 5-2 Changes in serum 25(OH)D concentrations in the low dose vitamin D treated group (LD) and in the high dose vitamin D treated group (HD) from baseline to 10wk ........................ 60 Figure 5-3 Baseline serum 25(OH)D concentrations according to ethnicity ................................ 61 Figure 5-4 Changes in mean serum PTH levels according to baseline 25(OH)D status in subjects in the high dose vitamin D treated group ...................................................................................... 63 vi Contributions The vitamin D concentrated casein premix was made in-house by me at the Food Science Research lab, Ryerson University, Toronto, Ontario. The fortified cheeses were made by Agropur Cooperative at the