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Investigation of vitamin D3 content in fortified fluid milk and the stability of vitamin D3 in milk to… Liu, Yang 2013

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INVESTIGATION OF VITAMIN D3 CONTENT IN FORTIFIED FLUID MILK AND THE STABILITY OF VITAMIN D3 IN MILK TO LIGHT EXPOSURE  by Yang Liu  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Food Science)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2013  © Yang Liu, 2013  Abstract  Fortified milk is the main dietary source of vitamin D, an important nutrient for skeletal health and reducing the risk of some chronic diseases. However, non-compliance of vitamin D fortification of fluid milk has been reported as a recurring problem. The purpose of this research is to analyze vitamin D contents of fortified fluid milk sold in Vancouver, the stability of commercial vitamin D formulations in milk upon light exposure during refrigerated storage, and the thermal stability of vitamin D2 and D3. Vitamin D content was determined for 104 fluid milk products purchased from the Vancouver retail market from October 2011 to September 2012. Fortification noncompliance was defined as vitamin D levels outside the range of 35.2-46.9 IU/100 mL, corresponding to regulatory requirements stated by the Canadian Food Inspection Agency. The results showed 54% under-fortification and 4% over-fortification with significant variation in vitamin D content among milk from different brands, with different fat content and sampled at different times. In particular, higher incidence of under-fortification was observed in skim milk samples. To investigate the stability of vitamin D3 to light exposure, skim milk was fortified with one of four vitamin D3 formulations along with vitamin A: crystalline vitamin D3, two water dispersible formulations and an emulsified vitamin A/D premix. Vitamin D loss after 22 days of storage at 4 °C with exposure to light of 2000 lux intensity ranged from 37% to 71% depending on the different formulation, and was accompanied by vitamin A loss. To investigate the effects of pH and heat on stability, vitamin D2 and D3 in citratephosphate buffer at pH 3.5 and 6.6 were heated at 72 °C for 15 seconds. Less than 8%  ii  vitamin D loss was observed. No significant difference was found in thermal stability between vitamin D2 and D3 or between the pH conditions during heating. These results indicate the possible approach to improve vitamin D fortification compliance in fluid milk by choosing a vitamin D formulation with improved dispersibility and greater stability against light exposure. Further research is needed to investigate other potential factors affecting the vitamin D content in fortified milk.  iii  Table of Contents  Abstract .................................................................................................................................... ii Table of Contents ................................................................................................................... iv List of Tables ........................................................................................................................ viii List of Figures .......................................................................................................................... x List of Abbreviations ............................................................................................................ xii Acknowledgements .............................................................................................................. xiii Dedication ............................................................................................................................. xiv Chapter 1: Introduction and literature review ................................................................... 1 1.1  Background ............................................................................................................... 1  1.2  Literature review ....................................................................................................... 2  1.2.1  General introduction of vitamin D ........................................................................ 2  1.2.2  Vitamin D status of Canadians ............................................................................. 8  1.2.3  Vitamin D fortification in milk ........................................................................... 10  1.2.3.1  Milk consumption and serum 25(OH)D ..................................................... 10  1.2.3.2  Non-compliance of vitamin D fortification ................................................ 13  1.2.3.3  Vitamin D fortification practices ................................................................ 17  1.2.4  Vitamin D stability.............................................................................................. 18  1.2.4.1  Stability of solid vitamin D ......................................................................... 18  1.2.4.2  Stability of vitamin D in foods.................................................................... 19  1.2.5  Encapsulation of fat soluble vitamins ................................................................. 25  1.2.6  Analysis of vitamin D content in food samples .................................................. 26  iv  1.2.6.1  Biological assays ......................................................................................... 26  1.2.6.2  Spectrophotometric methods ...................................................................... 28  1.2.6.3  HPLC methods ............................................................................................ 28  Chapter 2: Hypotheses and research outline..................................................................... 34 2.1  Hypotheses and objectives ...................................................................................... 34  Chapter 3: Vitamin D of fortified fluid milk in Vancouver ............................................. 36 3.1  Materials ................................................................................................................. 36  3.1.1  Chemicals and apparatus..................................................................................... 36  3.1.2  Vitamin standards ............................................................................................... 38  3.1.3  Sampling of commercially fortified fluid milks ................................................. 38  3.2  Vitamin D determination ........................................................................................ 39  3.2.1  Saponification ..................................................................................................... 41  3.2.2  Extraction ............................................................................................................ 41  3.2.3  Sample purification ............................................................................................. 42  3.2.4  HPLC analysis of vitamin D ............................................................................... 42  3.3  Statistical analysis ................................................................................................... 44  3.4  Results and discussion ............................................................................................ 44  3.4.1  Establishment of the adapted AOAC official method ........................................ 44  3.4.2  Vitamin D in commercially fortified fluid milk ................................................. 47  3.5  Conclusions ............................................................................................................. 55  Chapter 4: Vitamin D in laboratory fortified milk exposed to light ............................... 56 4.1 4.1.1  Materials ................................................................................................................. 58 Commercial Vitamin D Formulations ................................................................ 58  v  4.1.2  Preparation of fortified milks for the study of stability of vitamin D to light  exposure .......................................................................................................................... 58 4.1.3  Chemicals and apparatus for vitamin D and vitamin A determination ............... 60  4.2  Experimental set-up for the light exposure study ................................................... 62  4.3  Analysis of vitamin D and vitamin A ..................................................................... 62  4.4  Microbial total plate count ...................................................................................... 63  4.5  Statistical analysis ................................................................................................... 65  4.6  Results and discussion ............................................................................................ 65  4.7  Conclusion .............................................................................................................. 78  Chapter 5: Thermal stability of vitamin D2 and vitamin D3 solutions at pH 3.5 and 6.6 ................................................................................................................................................. 79 5.1  Materials ................................................................................................................. 79  5.2  Thermal processing ................................................................................................. 81  5.3  Vitamin D analysis .................................................................................................. 81  5.4  Statistical analysis ................................................................................................... 81  5.5  Results and discussion ............................................................................................ 82  5.6  Conclusion .............................................................................................................. 82  Chapter 6: General discussion and conclusions ................................................................ 85 References .............................................................................................................................. 94 Appendices ........................................................................................................................... 102 Appendix A Time and places for commercial milk sampling and analysis ..................... 102 Appendix B Vitamin D content of commercial fortified fluid milks and t-test for two containers of same product in each set. ............................................................................ 106  vi  Appendix C Vitamin formulations used in light exposure study. ..................................... 109  vii  List of Tables  Table 1.1 Dietary reference intakes of vitamin D by Health Canada. .................................. 5 Table 1.2 Vitamin D content in selected foods. .................................................................... 6 Table 1.3 Summary of surveys conducted for vitamin D content in fluid milk.................. 15 Table 1.4 Summary of studies on vitamin D stability in model system or foods. .............. 21 Table 1.5 Summary of AOAC HPLC methods for vitamin D in dairy products. ............... 30 Table 1.6 Solid phase extraction techniques for vitamin D determination. ........................ 33 Table 3.1 Overview of milk samples of seven brands. ....................................................... 40 Table 3.2  Flow rates and concentrations of mobile phase components for determination of  vitamin D in fluid milk by HPLC. .......................................................................................... 43 Table 3.3 Vitamin D3 content of seven brands of fluid milks collected in four sampling sets. ................................................................................................................................................. 51 Table 3.4 Results of three way ANOVA for vitamin D in commercial fluid milk. ............ 53 Table 4.1  Ingredients of vitamin formulations used in the present research....................... 59  Table 4.2  Flow rates and concentrations of mobile phase components for determination of  vitamin A in fluid milk by HPLC. .......................................................................................... 64 Table 4.3  Vitamin D3 content during 22 days of storage at 4 °C of milks fortified with four  different vitamin formulations. ............................................................................................... 66 Table 4.4 Results of three-way ANOVA for retention of vitamin D3 in fortified skimmed milk exposed to light. .............................................................................................................. 67 Table 4.5 Tukey's test of vitamin D retention of four formulations with different light exposure times. ....................................................................................................................... 70  viii  Table 4.6  Vitamin A content in skimmed milk fortified with vitamin A and D exposed to  light of 2000 lux or stored in the dark..................................................................................... 76 Table 4.7 Vitamin A content in skimmed milk fortified with vitamin A or vitamin A and D, and stored in the dark. ............................................................................................................. 77 Table 5.1  The impact of thermal treatment (72 °C for 15 seconds) on vitamin D content in  pH 3.5 and pH 6.6 buffers.. ..................................................................................................... 83 Table 5.2  The result of two-way ANOVA for vitamin D2 and D3 retention after thermal  treatment (72 °C for 15 seconds) at pH 3.5 or pH 6.6. ........................................................... 84 Table 6.1  Possible reasons contributing to the variation in vitamin D content in sampled  milk. ........................................................................................................................................ 87 Table 6.2  Comparison of vitamin D loss in milk exposed to light during storage............. 90  ix  List of Figures  Figure 1.1  Molecular structures of vitamin D2 (a) and vitamin D3 (b). ................................ 3  Figure 1.2 The impact of milk consumption on serum 25(OH)D of Canadians. ................. 11 Figure 1.3 Average serum 25(OH)D concentration versus the percentage of different age groups in Canada with high milk consumption. ..................................................................... 12 Figure 1.4  Degradation of solid vitamin D under different storage conditions. ................. 20  Figure 3.1  Overview of sampling of commercial milk products for vitamin D measurement.  ................................................................................................................................................. 37 Figure 3.2  HPLC chromatogram of vitamin D2 and vitamin D3 standards......................... 46  Figure 3.3 Standard curve of vitamin D3 determination using HPLC (vitamin D2 peak area ratio versus vitamin D3 concentration). .................................................................................. 48 Figure 3.4  Sequence of solvents used for washing and re-conditioning the analytical HPLC  column..................................................................................................................................... 49 Figure 3.5  Comparison of vitamin D3 content in skim milk versus milk with 2 or 3.25% fat.  Values shown are mean and standard deviation of four sets (n≥8)....................................... 52 Figure 4.1  Overview of study design to test stability of different vitamin D formulations in  milk during storage with light exposure. ................................................................................ 57 Figure 4.2  Set up for the exposure of fortified skimmed milk samples to light. ................ 61  Figure 4.3 Trends of percent retention of vitamin D3 content in skimmed milk fortified with four different formulations during 22-day storage at 4 °C with light exposure. ..................... 69 Figure 4.4 Vitamin D % retention in outside bottle (- O) and center bottle (- C) of skimmed milk fortified with four vitamin D3 formulations (n=3).......................................................... 72  x  Figure 4.5  Vitamin D retention as a function of the interaction of formulation and position  at different exposure times. ..................................................................................................... 73 Figure 4.6  Vitamin D retention as a function of the interaction of formulation and exposure  time at different positions. ...................................................................................................... 75 Figure 5.1  Overview of study design for stability of vitamin D2 and D3 to thermal treatment  in a model system.................................................................................................................... 80 Figure 6.1 The percentages of milk samples categorized as under-fortified, over-fortified or satisfying the fortification target for vitamin D (based on a 20% margin of tolerance) in the present study (2012) compared to the study by Chen et al. (1993). ....................................... 86  xi  List of Abbreviations  1, 25(OH)2D: 1α,25-Dihydroxyvitamin D 25(OH)D: 25-Hydroxyvitamin D AD premix: Emulsified mixture of vitamin D3 and vitamin A palmitate from Kingsway Chocolate AOAC: Association of Official Analytical Chemists BHT: Butylated Hydroxytoluene CFIA: Canadian Food Inspection Agency Crystalline D3: Food grade crystalline vitamin D3 from DSM nutrition CWS D3: Dry vitamin D3 100 CWS/AM from DSM nutrition FTIR: Fourier-Transform Infrared HPLC: High Performance Liquid Chromatography HTST: High Temperature Short Time IOM: Institute of Medicine IU: International Unit RDA: Recommended Dietary Allowance SDS D3: Dry vitamin D3 100 SD/S from DSM nutrition SPE: Solid Phase Extraction UHT: Ultra High Temperature UVB: Ultraviolet B  xii  Acknowledgements  This research would not have been completed without the support from many people. I owe my deepest gratitude to my supervisor, Dr. Eunice Li-Chan, who gave me the opportunity to work in her lab and provided the kind guidance and support through my research. I would like to thank my committee members, Dr. Christine Scaman and Dr. Tim Green, who gave me valuable advice on my research. I would also like to acknowledge the UBC Food Nutrition and Health Vitamin Research Fund, which made this research possible. I want to thank all my lab colleagues, especially Benny Chan and Imelda Cheung. Benny developed the HPLC method in this research and worked together with me to analyze the milk samples. Imelda provided me guidance on almost everything in our lab. I also want to show my gratitude to Dr. Ronaldo Cerri, who kindly collected the raw milk for this research from UBC Dairy Education and Research Center. I also want to thank Ana Cancarevic, Valerie Skura and Pedro Aloise for their advice and assistance on my experiments. Without the help of Dr. Gary Sandberg and Mr. Kim Cummings and the equipment at BCIT, I would not have been able to conduct the pasteurization of milk. Finally I would like to thank my husband and my dear friend Isabelle Lacroix. Without their ongoing support and encouragement, this research would not have been completed.  xiii  Dedication  This thesis is dedicated to my parents and my husband for their unconditional love and support As well as my dear friend Isabelle Lacroix for her precious friendship  xiv  Chapter 1: Introduction and literature review 1.1  Background The importance of vitamin D has increased in the past few decades as emerging  studies show the sunshine vitamin provides many other health benefits besides the wellknown contribution to skeletal health. The deficiency of vitamin D may raise the risks of some chronic diseases such as type II diabetes, autoimmune disorders, cardiovascular disease, and certain types of cancer (Holick, 2004; Vatanparast et al., 2010; IOM, 2011). Vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol) are two natural forms of vitamin D. Vitamin D2 is obtained from irradiation of plants or yeasts. Vitamin D3 is naturally occurring in some foods. In the human body, vitamin D3 can be synthesized in skin via exposure to sunlight. Skin pigment, latitude, seasons, clothing, amount of skin exposure and the use of sunscreen cosmetics are main factors affecting the endogenous vitamin D level (Calvo & Whiting, 2003; IOM, 2011). The concern of skin cancer is a reason to avoid long exposure to sunlight. The prevalence of vitamin D insufficiency and deficiency has long been a worldwide concern, especially in high risk groups such as young children, pregnant women and elderly people. These problems have been observed even in sun drenched areas, such as southern Europe, the Middle East, India, Africa and Australia (Mithal et al., 2009; Lips, 2010). The prevalence of vitamin D insufficiency in Canada, especially in dark skinned people living in northern latitudes has been reported (Cole et al., 2001; Rucker et al., 2002). In North America, food fortification and dietary supplements are the main approaches to ensure adequate vitamin D intake (Calvo et al., 2004). However, the study  1  of Whiting et al. (2007) showed that despite the fortification, the vitamin D intakes in Canada are not adequate to prevent vitamin D insufficiency. Non-compliance of vitamin D fortification that has been reported in several studies (e.g., Holick et al., 1992; Blank et al., 1995; Murphy et al., 2001) might be one of the reasons behind the vitamin D insufficiency, since consumers might access less or more vitamin D than what is being claimed on the labels of fortified foods. These previous studies did not indicate what caused the fortification non-compliance and whether or not the vitamin stability played a role in these cases. To understand the possible reasons of vitamin D non-compliance, it is important to investigate the content as well as stability of vitamin D in fluid milk. 1.2 1.2.1  Literature review General introduction of vitamin D Vitamin D refers to a group of secosteroid compounds with antirachitic activity.  Cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2) are two natural forms of vitamin D. In the human body, upon UVB irradiation, the 7-dehydrocholesterol in skin is first converted to previtamin D3 in which the vitamin precursor B-ring is open, then subsequently isomerized to vitamin D3. Similarly, vitamin D2 is formed via the conversion of ergosterol to previtamin D2 by UVB irradiation; this process mainly occurs in certain fungi or yeasts. The molecular structures of vitamins D2 and D3 are shown in Figure 1.1. The research of vitamin D has a long history. According to Norman & Henry (2007), Hirsch and Palm first associated the disease rickets with the lack of sunlight near the end of 19th century. In 1922, McCollum discovered a compound in cod-liver oil with antirachitic activity and named it vitamin D (Norman & Henry, 2007).  2  (a)  Figure 1.1  (b)  Molecular structures of vitamin D2 (a) and vitamin D3 (b).  The figure for vitamin D3 is adapted from Cholecalciferol in Wikipedia (retrieved March 19, 2011, from http://en.wikipedia.org/wiki/Cholecalciferol, Copyright Public Domain). The figure for vitamin D2 is adapted from Ergocalciferol in Wikipedia (retrieved March 19, 2011, from http://en.wikipedia.org/wiki/Ergocalciferol, Copyright Public Domain).  3  Vitamin D modulates calcium and phosphorus absorption, which is necessary for the health of bones and teeth. Vitamin D had been considered as the active form for healthy bone development until the discovery of its metabolites, 25-hydroxyvitamin D (25(OH)D) and 1, 25-dihydroxyvitamin D (1, 25(OH)2D). After synthesis in skin or intake from diet, vitamin D is converted into 25(OH)D in the liver, and then further transformation to 1, 25(OH)2D is completed in the kidney. The 25(OH)D is the main circulating metabolite while 1, 25(OH)2D is the active metabolite of vitamin D (Norman & Henry, 2007). At the early stage, scientists mainly focused on the function of vitamin D to develop healthy bones. Recent studies showed that the benefits of vitamin D are not limited to bone health. As a hormone, 1, 25(OH)2D was also found in different tissues such as intestine, kidney, liver, lung, brain, breast, cancer cells, muscle, and ovary (Norman & Henry, 2007). It is believed to modulate the immune system, associate with neuropsychological development of infants and reduce the risk of diabetes, cardiovascular diseases, and certain cancers (Holick, 2004; Vatanparast et al., 2010; IOM, 2011). However, excessive intake of vitamin D is dangerous. High dose of vitamin D may lead to weight loss, polyuria, heart arrhythmias, raised blood levels of calcium and result in vascular and tissue calcification, damage to the heart, blood vessels and kidneys (IOM, 2011). Table 1.1 shows the recommended dietary allowance and upper tolerance of vitamin D, as revised by Health Canada in 2011. The vitamin D level in selected foods can be found in Table 1.2.  4  Table 1.1  Dietary reference intakes of vitamin D by Health Canada.  Age group  Recommended dietary  Tolerable upper intake  allowance per day (RDA)  level per day  Infants 0-6 months  400 IU (10 μg)  1000 IU (25 μg)  Infants 7-12 months  400 IU (10 μg)  1500 IU (38 μg)  Children 1-3 years  600 IU (15 μg)  2500 IU (63 μg)  Children 4-8 years  600 IU (15 μg)  3000 IU (75 μg)  Children and adults 9-70 years 600 IU (15 μg)  4000 IU (100 μg)  Adults > 70 years  800 IU (20 μg)  4000 IU (100 μg)  Pregnancy & lactation  600 IU (15 μg)  4000 IU (100 μg)  Source: Health Canada, 2011a  5  Table 1.2  Vitamin D content in selected foods. IU per serving  % RDA  Cod liver oil, 1 tablespoon  1,360  227  Salmon (sockeye), cooked, 3 ounces  794  132  Mackerel, cooked, 3 ounces  388  65  Tuna fish, canned in water, drained, 3 ounces  154  26  Liver, beef, cooked, 3.5 ounces  46  8  Egg, 1 whole (vitamin D in yolk)  25  4  Milk, vitamin D-fortified, 1 cup  90  15  Orange juice fortified with vitamin D, 1 cup  100  17  Yeast-leavened bakery products, 100 g  90  15  Margarine, fortified, 1 tablespoon  72  12  Infant formula, 100 kcal  100  25  Formulated liquid diets, 1,000 kcal (2,500 kcal/day)  800  133  a  Natural foods b  Fortified foods c  a  The RDA of 600 IU for people from 1 to 70 years old was used for the calculation of %  RDA in all food items except infant formula, for which 400 IU was used. b  U.S. Department of Agriculture, Agricultural Research Service (2012).  c  Food and Drug Regulations (2012) and Health Canada (2004, 2006 and 2011b)  6  In Canada, vitamin D fortification is mandatory for fluid milk and milk powder, fluid goat milk and powder, margarine and other butter substitutes, infant formula and formulated liquid diet, foods used in very low energy diet, whole egg products and yolk products (Food and Drug Regulations, 2012). Health Canada (2006 & 2011b) also issued interim marketing authorizations for vitamin D addition to orange juice and yeastleavened bakery products. Both vitamin D2 and D3 can be used for food fortification. Published studies have not shown consistent conclusions on the potency of these two forms of vitamin D. Vitamin D2 was initially recognized as being equivalent to vitamin D3 although research revealed vitamin D2 was significantly less potent than vitamin D3 in some animals (Norman & Henry, 2007). The development of methods using serum 25(OH)D as a vitamin D biomarker provided an alternative to animal bioassays for the study of vitamin D potency. Trang et al. (1998) compared the ability of vitamin D2 and D3 to elevate serum 25(OH)D and reported that vitamin D3 increased serum 25(OH)D 1.7 times as much as vitamin D2 did at an intake level of 4,000 IU/day for two weeks. Armas et al. (2004) studied the effect of a single dose of 50,000 IU vitamin D2 or vitamin D3 on serum 25(OH)D over four weeks. They reported that the two forms maintained the serum 25(OH)D at a similar level during the first three days. However, the serum 25(OH)D dropped rapidly in the vitamin D2 group and was close to the baseline at day 14 while the serum 25(OH)D content in the vitamin D3 group reached a peak at that time. The vitamin D2 group showed a serum 25(OH)D content even lower than the control group at the end of the trial. As a response to the conclusion of Trang et al. (1998) and Armas et al. (2004), Holick et al. (2008) claimed,  based on their study, that there was no significant  7  difference between vitamin D2 and D3 to increase the circulating 25(OH)D. They divided the subjects into three groups to receive 1000 IU/day of either vitamin D2 or vitamin D3 or a combination for 11 weeks. By the end of the experiment, all of the subjects in the three groups showed an increase in serum 25(OH)D of 8-10 ng/mL. On the other hand, Heaney et al. (2011) provided more evidence in favour of vitamin D3 being more potent by comparing circulating 25(OH)D and fat storage of the subjects who were given the two vitamin D forms. Different methods and dosages were applied in these studies, which makes the comparison between studies difficult. The potency of vitamin D2 and D3 is still a controversial topic that remains unsettled, despite more evidence showing that vitamin D3 might be more effective than vitamin D2. More research needs to be conducted to establish the potency of vitamin D2 versus D3. 1.2.2  Vitamin D status of Canadians The importance of vitamin D has increased since more and more research  indicates that vitamin D provides multiple health benefits. In contrast to the increased awareness of the nutritional functions, low vitamin D status has long been a worldwide problem and needs to be addressed. Currently, the level of serum 25(OH)D is widely accepted for use as a biomarker to evaluate the status of vitamin D in the human body since it represents both skin synthesis and dietary intake of vitamin D (Calvo & Whiting , 2003; Langlois et al., 2010). However, various cut-offs of serum 25(OH)D level are being applied to define different vitamin D status in published studies. For example, serum 25(OH)D ranging from 27.5 nmol/L to 37.5 nmol/L have been used to define vitamin D deficiency based on  8  different health outcomes (Mithal et al., 2009; Langlois et al., 2010). The reported optimal level of serum 25(OH)D ranges from 40 nmol/L to 125 nmol/L in various studies (Rucker et al., 2002; Whiting et al., 2007; Lips, 2010; IOM, 2011). Recently, more studies agree that serum 25(OH)D level above 75 nmol/L is desirable for overall health (Mithal et al., 2009; Lips, 2010). There is no central organization that is responsible to establish the clinical standard of vitamin D deficiency, insufficiency and adequacy. In Canada, IOM (2011) suggests that adults with 25(OH)D levels below 30 nmol/L are considered at risk of poor bone health and that a concentration above 50 nmol/L is sufficient for most persons. A national survey was conducted between 2007 to 2009 to obtain health information of Canadians (Langlois et al., 2010). The cut-offs of 27.5 nmol/L and 37.5 nmol/L for vitamin D deficiency and insufficiency respectively, were applied in this survey. Considering these concentrations were lower than those suggested in the IOM 2011 report, the prevalence of vitamin D deficiency and insufficiency might be underestimated. According to the results of this survey, 4.1% of the population were deficient in vitamin D and the percentage insufficient was only 10%. However, if using 75 nmol/mL as the optimal vitamin D level cut-point, then only one-third of the population had sufficient 25(OH)D.  The authors did not report the percentage of  population having serum 25(OH)D less than the IOM insufficiency cut-off of 50 nmol/L. Rucker et al. (2002) reported that more than one third of western Canadians have 25(OH)D less than 40 nmol/L in winter, spring and fall. Cole et al. (2001) indicated that more than 20% of young Canadian women have low vitamin D status (less than 40 nmol/L). Vitamin D status might be affected by many factors such as skin colour,  9  latitude, season, dietary and supplementary intake. In North America, fortified foods and supplements are the main sources of vitamin D during seasons with limited sunlight (Calvo et al., 2004). Based on the 2011 report of IOM, Health Canada (2011a) increased the recommended intake of vitamin D from 200 IU to 600 IU for people aged 1 to 70. Daily intake of 400 IU is considered adequate for infants while people over 70 need to consume 800 IU. 1.2.3 1.2.3.1  Vitamin D fortification in milk Milk consumption and serum 25(OH)D In Canada, fortification of milk with vitamin D is mandatory. As a result, milk is  the main dietary vehicle of vitamin D for Canadians, followed in descending order by "meat and meat alternatives, other foods, grain products, vegetable and fruit" (Vatanparast et al., 2010). Vitamin D intake via milk consumption was reported to contribute 49% of the total dietary vitamin D (Vatanparast et al., 2010). The 2007 to 2009 Canadian Health Measures survey (Langlois, 2010) indicated that consumption of milk was a significant factor contributing to the increase of serum 25(OH)D concentration (Figure 1.2). The mean serum 25(OH)D of people who consumed milk more than once per day was 75.0 nmol/L while those who drank milk less than once per day had mean serum level of 62.7 nmol/L. The average serum 25(OH)D of different age groups are plotted against the percentage of that population group drinking milk more than once per day in Figure 1.3. The proportion of high frequency of milk consumption declined with increasing age to  10  Plasma 25(OH)D (nmol/L)  76 74 72 70 68 66 64 62 60 58 56 More than once a day  Once a day  Less than once a day  Milk consumption Figure 1.2 The impact of milk consumption on serum 25(OH)D of Canadians. Based on data of national survey conducted between 2007 and 2009 (Langlois et al., 2010).  11  70  74  60 50  72  40 70 30 68  20  66  10  64  0 6-11 25(OH)D  Figure 1.3  12-19  20-39  40-59  % of samples  25(OH)D nmol/L  76  60-79 yrs  milk consumption more than once per day  Average serum 25(OH)D concentration versus the percentage of different  age groups in Canada with high milk consumption. (Based on the data from the 2007-2009 Canada Health Measures Survey, as reported by Langlois et al., 2010).  12  40-59 years and then increased slightly in the 60-79 year old group. The serum 25(OH)D was distributed in a U shape from the young group to senior group. Although the distribution of serum 25(OH)D did not follow the exact pattern of high frequency milk drinkers, the trends were similar for the groups between 6-39 years of age. For different age groups, the serum 25(OH)D levels of those drinking milk more than once per day were all above 70 nmol/L despite the age difference (Langlois et al., 2010). 1.2.3.2  Non-compliance of vitamin D fortification Vitamin D is classified as risk category B nutrient by Health Canada, which  causes serious adverse effects at high intake level, but with low risk of excessive intake at the proposed level of addition for discretionary fortification (Health Canada, 2005). In 1991, over-fortification of vitamin D in home-delivery milk in Boston resulted in 41 patients being hospitalized and 2 deaths (Blank et al., 1995). Jacobus et al. (1992) reported the analysis of milk products being associated with the 1991 hypervitaminosis D cases in Boston. The result showed that none of the products contained vitamin D at the required level of 400 IU/quart. In this over-fortification incident, the vitamin D concentration ranged from less than 40 IU/quart to more than 200,000 IU/quart in fortified products. Half of the samples were fortified with a concentration at 73-580 times that of the required level. Furthermore, vitamin D levels varied greatly in milk sampled in April and June 1991. For example, homogenized whole milk from a Boston local dairy contained over 200,000 IU vitamin D per quart in April, while in the follow up test in June, vitamin D was found at a level of less than 40 IU/quart in this product. The investigation also found the concentrate used for milk fortification was labelled vitamin D2 while analysis revealed it contained vitamin D3.  13  The fortification non-compliance of vitamin D has been a recurring problem in milk products and not limited to North America. Table 1.3 summarizes the findings of several surveys on vitamin D content in fluid milk in the past 25 years. Tanner et al. (1988) summarized the results of two surveys on vitamin D content of fluid milk conducted in Oregon and Ohio states, US. In the Oregon survey, 449 milks and milk products were analyzed during 1979-1983. In the Ohio survey, 23 milk products were determined without indicating analysis dates. A general trend was found in these two surveys, which was lower vitamin D content in milk of lower fat content. For example, in the Oregon survey, the percentage of milk containing vitamin D at less than 50% of label value was 28% for the whole milk and 47% for the skim milk. In the Ohio survey, all whole milk samples contained vitamin D at a level more than 50% of the claimed amount while 37.5% of the skim milk contained less than half of the label amount. Tanner et al. (1988) indicated vitamin addition before fat separation might be the reason for lower fat soluble vitamin in milk with lower fat content. Holick et al. (1992) examined vitamin D content in 42 milk samples of 13 brands and 10 infant formula samples of 5 brands from five Eastern states in United States. The results showed that more than 60% of the milk samples were under-fortified and only 29% of the milk samples contained 80 -120 % of the declared label amount. More than 30% of the 42 samples contained vitamin D less than 50% of the label value. Three out of 14 skim milk samples contained vitamin D lower than the detection limit of 0.5 IU/100 mL. One sample contained vitamin D3 despite the fact that vitamin D2 was stated on the label. All 10 infant formula samples were over-fortified with vitamin D and 70% of the  14  Table 1.3  Summary of surveys conducted for vitamin D content in fluid milk.  Reference  Location  Sample size  Under-fortification  Over-fortification  Standard of compliance  Tanner et al., 1988  US  539  53%  23%  33.8 - 50.7 IU/100 mL  Holick et al., 1992  US  42  62%  10%  33.8 - 50.7 IU/100 mL  Chen et al., 1993  US  79  51%  29%  33.8 - 50.7 IU/100 mL  BC, Canada  15  27%  47%  28.8 - 43.2 IU/100 mL  Faulkner et al., 2000  ON, Canada  45  36%  38%  31.7 - 51.6 IU/100 mL  Murphy et al., 2001  US  648  28%  25%  33.8 - 50.7 IU/100 mL  Thomson, 2006*  New Zealand  18  28%  39%  Various label claim  Laleye et al., 2009  United Arab Emirates 54  31%  30%  32 - 48 IU/100 mL  19%  41%  42.3 - 52.9 IU/100 mL  Patterson et al., 2010 US  120  * The percentages for the study by Thomson (2006) were based on all fortified foods investigated including fluid milk.  15  samples contained more than 200% of the labelled amount and the highest was overfortified with more than four times the declared vitamin D content. In a follow-up study (Chen et al., 1993), the research group of Holick, Chen and colleagues analyzed 79 milk samples from United States and 15 samples from British Columbia (BC), Canada. The fortification situation was not improved in the United States with 51% of the samples containing less than 80% of the claimed vitamin D amount and 29% samples being fortified with over 120% of the claimed amount. The samples from BC also showed fortification non-compliance, with 47% over-fortified samples and 27% under-fortified samples. Faulkner et al. (2000) reported an investigation on fortified fluid milk in Ontario. Whole milk, 2% and skim milk from 15 brands were analyzed in this research. More than 70% of the 45 samples were either under or over-fortified. Vitamin D was not detected in four products including one 2%, one skim and two whole milk products. Whole milk and skim milk showed similar patterns, with almost 50% over-fortification and 30% underfortification. The fortification compliance in 2% milk was more acceptable, with close to 50% of the samples satisfying the standard of 31.7 - 51.6 IU/100 mL. The criteria of acceptance applied in this research was wider than the CFIA (2012) requirement of 35.2 46.9 IU/100mL, thus the non-compliance might be even worse if applying the more strict CFIA range. Murphy et al. (2001) evaluated the fortification of fluid milk in New York states. Whole milk, 1%, 2% and skim milk were sampled over four years for vitamin D determination. More than half of the 648 samples showed fortification non-compliance.  16  The Institute of Environmental Science and Research Limited conducted a survey on vitamin A, D and calcium contents in the fortified foods of New Zealand (Thomson, 2006). Eighteen samples including milk, margarine, food drink, and baby food were analyzed for vitamin D content. Overages of 25-70% were observed in 39% of the samples and 28% of the samples were under-fortified with vitamin D levels 47-68% less than the label claim. Laleye et al. (2009) sampled whole milk, low fat milk and skim milk in United Arabs Emirates and found 31% of the 54 samples were under-fortified and 30% were over-fortified. In a recent investigation, Patterson et al. (2010) analyzed 120 milk products sampled across the US. Wide variation was observed with vitamin D content ranging from undetectable to almost 200% of the label value. 1.2.3.3  Vitamin D fortification practices The conditions of vitamin storage and addition might affect the final fortification  level. In a survey conducted in North Carolina (Hicks et al., 1996), 54% of the surveyed dairy manufacturers stored vitamin D at ambient temperatures despite the suppliers' recommendation for refrigerated storage. The 13 manufacturers added the vitamin at 9 different points during processing and most of them added vitamin preparation before pasteurization. This survey divided the dairy processors into two groups based on the method of vitamin addition. For those who used injection meter, seven processors added vitamin D at five different points after standardization and before homogenization. Two processors injected the vitamin D after homogenization and before the pasteurization. When using batch addition method, three processors added vitamin D before milk fat separation, and four processors added vitamin D after standardization and before  17  homogenization. This survey indicated possible losses of vitamin D during the storage and processing. The researchers also suggested that similar fortification practices might be used nationwide in the United States. No information of the actual vitamin D addition practices in Canada was found, although CFIA does provide detailed guidance on the specific procedures and protocols for vitamin addition to dairy products (CFIA, 2012). In the Dairy Products Inspection Manual, CFIA (2012) provides equations to calculate the amount of vitamin D for batch addition and injection of continuous processing. CFIA (2012) also suggests that vitamin D can be added at different points in the processing system, from batch pasteurizer or HTST pasteurizer balance tank to finished powder prior to final packaging. 1.2.4 1.2.4.1  Vitamin D stability Stability of solid vitamin D Huber and Barlow (1943) indicated that all forms of vitamin D were susceptible  to oxidation, and more rapidly oxidized under dry condition than in emulsions. Byrn (1976) reported in his study of solid-state reactions of drugs that crystalline vitamin D2 was sensitive to light, air, and heat and yielded several decomposed products whose structures were not determined. The crystalline vitamin D2 was observed to have completely turned to yellow after six months at ambient temperature with free access to air, and the elemental composition was changed from C28H44O to C28H44O8. The crystalline vitamin D2 was fully degraded when heated at 80 ºC no matter whether light was present or not but was stable at this high temperature for one week if there was no contact with oxygen.  18  In their accelerated degradation study, Kozlov et al. (1977) observed changes in the infrared spectrum in terms of the shape and intensity of bands around 890 cm-1 and 970 cm-1, which they attributed to the breakdown of the conjugated system caused by oxidation. Ketones were also generated during the oxidation and showed bands at 1660 cm-1 and 1715 cm-1. Grady and Thakker (1980) compared the stability of vitamin D2 and D3 powders under four different storage conditions (Figure 1.4). When stored in a desiccator and in the presence of oxygen, less than 5% vitamin D2 was left while 99% of vitamin D3 was maintained after 56 days. Vitamin D3 became more sensitive to the temperature when humidity was increased. The stability of vitamin D2 was greatly improved at 85% relative humidity and room temperature. When the environment temperature increased from 25 ºC to 40 ºC, the degradation of both forms was accelerated. At room temperature both forms were stable in nitrogen during 56 days storage. The authors also indicated that the decomposed products of vitamin D2 possessed higher polarity. DeRitter (1982) indicated that vitamin D3 was also sensitive to trace metals. Stewart et al. (1984) reported that under room temperature in the presence of light, the crystalline vitamin D2 would be gradually degraded to an orange colour powder in 6 months, which confirmed the observation of Byrn (1976). The oxidation resulted in the destruction of the triene structure. 1.2.4.2  Stability of vitamin D in foods It is reported that vitamin D is generally stable during food processing and storage  (Kazmi et al., 2007; Hanson & Metzger, 2010). However, vitamin D loss was reported in some other research. Table 1.4 summarizes the studies of vitamin D stability in foods.  19  120  % Remaining  100 80 60 40  20 0 0  10  20  30  40  50  60  Days  Figure 1.4  D2 in desiccator, 25 ºC  D3 in desiccator, 25 ºC  D2 45% RH, 40 ºC  D3 45% RH, 40 ºC  D2 85% RH, 25 ºC  D3 85% RH, 25 ºC  D2 85% RH, 40 ºC  D3 85% RH, 40 ºC  Degradation of solid vitamin D under different storage conditions.  (Based on data of Grady and Thakker, 1980).  20  Table 1.4  Summary of studies on vitamin D stability in model system or foods.  Analyte  Result  Reference  Vitamin D3 in acetonitrile  60% loss by light exposure and  Renken &  storage at 21 °C after 10 days  Warthesen, 1993  Vitamin D3 in skim milk  25% loss by light exposure in 1.8 L plastic container after 10 days  Vitamin D3 in spray-dried  No loss during processing  Indyk et al., 1996  Vitamin D2 in model system  Vitamin D oxidation caused by  King & Min,  (12% H2O+88% acetone)  riboflavin + light  1998  Vitamin D3 in fish, eggs, &  Less than 10 % loss during  Mattila et al.,  vitamin D2 in mushrooms  household cooking  1999  Vitamin D3 in orange juice  Stable for 30 days storage  Tangpricha et al.,  milk powder  2003 Vitamin D3 in cheese,  Stable during processing and cold  Kazmi et al.,  yogurt, & ice cream  storage  2007  Vitamin D3 in UHT whole  20 -57% loss caused by light during  Saffert et al.,  milk  12 weeks storage  2008  Vitamin D3 in UHT low fat  35-65% loss induced by light during  Saffert et al.,  milk  12 weeks storage  2009  21  Renken and Warthesen (1993) studied the impact of light, air, storage temperature on the stability of vitamin D3 in an acetonitrile model system and skim milk. They exposed the acetonitrile solution of vitamin D3 to fluorescent light of 2,260 lux (210 footcandle) and air was bubbled into samples by a pump. Two storage temperatures, 4 °C and 21 °C, were applied. In the acetonitrile model system, the impact of light exposure was not significant on vitamin D3 stability. Air contributed the greatest loss of vitamin D3, while the combination of air and high storage temperature also caused significant loss of vitamin D. In skim milk, the light exposure alone contributed the greatest vitamin D3 loss, followed by air with light and air alone. In a 1.8 L plastic container, the vitamin D3 level in skim milk was decreased by 25% after being exposed to 3229 lux (300 footcandle) for 10 days. The difference between acetonitrile model system and skim milk might be caused by different ways of aeration. Air was flushed into the headspace of skim milk while being bubbled through the acetonitrile solution. Renken and Warthesen (1993) assumed that the photo-degradation of vitamin D was caused by the singlet oxygen. This hypothesis was confirmed by later research (King & Min, 1998 & 2002; Li & Min, 1998). Singlet oxygen is produced by light exposure and has higher energy than regular oxygen. It may react with susceptible bonds in vitamin D and cause degradation. Renken and Warthesen (1993) also noticed the stratification of vitamin D3 in milk with more vitamin D3 in the top layer. In Renken and Warthesen's research, light was a significant factor for vitamin D loss in skim milk while not in acetonitrile model system. This might be caused by the presence of riboflavin in milk. As a photo-sensitizer, riboflavin can trigger the formation of singlet oxygen, which may cause degradation of vitamin D. King and Min (1998)  22  tested the stability of vitamin D2 with or without riboflavin in a model system of 12% distilled water and 88% acetone. Instead of vitamin D2 measurement, oxygen loss in the headspace of sealed sample container was analyzed to confirm the oxidation of vitamin D2. It was based on the following reaction: singlet oxygen was produced by a sensitizer (in this research, riboflavin), then reacted with a compound to be depleted from the headspace. Similar to the result of Renken and Warthesen (1993), no oxidation was found in sole vitamin D2 solution, even though it was exposed to light of 4,000 lux. In the absence of riboflavin, no singlet oxygen was produced and triplet oxygen could not react with vitamin D2. Oxygen loss was also not found in the sample with riboflavin but stored in the dark since riboflavin was not excited. King and Min (2002) further explored the mechanism using multiple instruments. Their result of high performance liquid chromatography (HPLC) showed that two extra peaks appeared in the chromatogram of the vitamin D solution containing riboflavin on light exposure, that were not found in other samples. UV scan confirmed the reaction occurred on the triene ring of vitamin D2. The mass spectrum showed that the mass of vitamin D2 was increased by one oxygen atom. Hydroxyl group was presented in oxidized vitamin D2 while no carbonyl group was found by IR spectrum. Based on the results of different instrumental analysis, King and Min (2002) concluded that oxygen reacted with the triene ring of vitamin D2 and a 5, 6epoxide was formed. Indyk et al. (1996) studied stability of vitamin D3 in milk during spray-drying. Fortified milk went through low pressure preheating, direct steam injection at 95 °C, evaporation and spray-drying at 149 °C and fluid bed finish at 107 °C. No significant change of vitamin D content was found after the processing. Thus the common practice  23  of 30% overage addition of vitamin D to ensure the final fortification level was not necessary. Mattila et al. (1999) studied the stability of vitamin D3 found naturally in egg, fish and vitamin D2 in wild mushroom and claimed that vitamin D was generally stable. The losses during the storage and thermal processing of these foods were less than 10%. Tangpricha et al. (2003) reported vitamin D3 in fortified orange juice was stable during 30 days storage at 4 ºC. Kazmi et al. (2007) studied the stability of vitamin D3 in crystalline and emulsion forms during processing and storage of yogurt, ice cream and cheese. The acidification of yogurt and aeration of ice cream showed little impact on the vitamin D 3 content. No significant difference was found in the loss of the two forms of vitamin D3. The vitamin D3 losses subsequent to processing were 3% and 1% in yogurt and ice cream respectively. Pike and Brown (1984) indicated that vitamin D was not stable in acidic media and the instability increased with increased temperature. Whether the low temperature contributes to the low vitamin D loss during production and storage of yogurt was still unknown. Vitamin D3 loss of 3-5% was observed during cheese making. Of the vitamin D3 recovered, about 7-9% was found in whey. The crystalline form showed slightly higher loss than the emulsified one, with more vitamin D3 found in whey for the product fortified with crystalline form. Saffert et al. (2008) observed vitamin D3 loss in ultrahigh temperature (UHT) pasteurized whole milk stored in clear or pigmented bottles at 23 ºC. More than half of the vitamin D3 content in milk was lost after 12 weeks storage in clear bottles. Most samples lost at least 20% of vitamin D3 content despite the protection provided by  24  pigmented bottles. In low fat UHT milk the vitamin D3 was more sensitive, with a loss up to 65% of the original content (Saffert et al., 2009). 1.2.5  Encapsulation of fat soluble vitamins Encapsulation technology provides many benefits to the food fortification.  Sensitive ingredients can be protected by encapsulation matrix from oxygen and light. Encapsulation may change the physical properties of food ingredients to improve the quality of fortified products, for example, reducing the size of particles or droplets to achieve a more homogeneous dispersion of nutrients in final products. Liquid agents can be converted into solid particles and fat soluble ingredients might be added into an aqueous system after encapsulation. Fat soluble substances are often dissolved in vegetable oil. An emulsion is then formed by dispersing the oil droplets into an aqueous protein or polysaccharide solution. The last step is either spray drying or freeze drying to produce a free-flowing powder (Palzer, 2009). Milk proteins can be used as encapsulating materials for fat soluble nutrients. Forrest et al. (2005) investigated the interactions between β-lactoglobulin A, β-casein and vitamin D3. Both proteins showed strong ability to bind vitamin D3. β-Lactoglobulin is able to bind a variety of lipophilic compounds, thus is considered as a suitable natural vehicle for fat soluble nutrient delivery. Forrest et al. (2005) suggested that βlactoglobulin A might provide protection to vitamin D3 during fermentation since the binding between the two compounds was stable from pH 8.0 to 2.5. Ron et al. (2010) reported nano-encapsulation of vitamin D2 for the fortification of clear beverages. Lowest turbidity was achieved by adjusting pectin concentration in a β-lactoglobulin-vitamin D2  25  solution at pH 4.25. Semo et al. (2007) encapsulated vitamin D2 within casein micelles and suggested that this nano-vehicle could provide partial protection against the UV light induced degradation of vitamin D2. Modified starch, corn starch, vegetable oil, acacia gum, and sucrose might be used in commercial formulas of vitamin D (DSM, Netherlands). Disaccharides can be included in the matrix to improve the retention of characteristics of the substance of interest(Sosa et al., 2011). Sucrose has been used to form coating matrix with other compounds for various oil products because of its good solubility in water, low cost and long shelf-life (Kaushik & Roos, 2006). Encapsulation of fish oil also involves sucrose in coating matrix since sucrose can develop cross-linking by Maillard reactions and thus improve the protection from oxygen (Beindorff & Zuidam, 2010). Silicon oxides, aluminum oxides or tripolyphosphate can be used alone or combined during encapsulation of active compounds. These chemicals are believed to improve the barrier ability of coatings, thus protect active compounds from oxygen, water vapor, and flavour permeation (AmbergSchwab et al., 2006; Desai & Park, 2006). 1.2.6  Analysis of vitamin D content in food samples The analysis of the vitamin D content in foods is important to ensure that amounts  claimed on labels are met. Different types of methods have been developed and improved for vitamin D determination. 1.2.6.1  Biological assays The earliest official methods for vitamin D determination were bioassays, in  which the bioactivity of vitamin D was determined by its ability to prevent or improve deficiency symptoms in vivo (Otles & Karaibrahimoglu, 2005). Rat line test was the first  26  official method to measure vitamin D content in foods or pharmaceutical products (Norman & Henry, 2007). According to AOAC method 936.14 (Cunniff, 1995), rachitic rats are fed with foods containing different amounts of vitamin D for the establishment of standards. After seven days, the bone of rats are processed with a silver nitrate solution. The newly deposited calcium in bone stains dark. Standards are set up by associating the dark regions on bone to the vitamin D amount in diet. Vitamin D content in sample is determined by comparing the dark region with standards. This method does not distinguish vitamin D2 from vitamin D3. To overcome this disadvantage, chick assay was introduced. Vitamin D3 shows 10 times greater potency than vitamin D2 in the chick, thus can be distinguished from vitamin D2 (Norman & Henry, 2007). It involves setting up standards by feeding newborn chicks with fortified feed containing different levels of vitamin D. The percentage of bone ash is determined after three weeks on the diet. Other bioassays include in vivo and in vitro intestinal calcium absorption, which investigate the ability of the test compound to stimulate the absorption of calcium in the small intestine. In these assays,  45  Ca2+ is used to indirectly quantify vitamin D content  (Norman & Henry, 2007). Bioassays can detect vitamin D at very low concentration. For example, the rat line test can detect as low as 12 ng of vitamin D. However, bioassays require long preparation times, which make them less convenient as a routine analysis method compared to other methods. The sacrifice of animals is also against the increasing awareness of animal welfare.  27  1.2.6.2  Spectrophotometric methods IR spectroscopy has been used for qualitative analysis of vitamin D. In solid  forms, vitamin D2 can be distinguished from vitamin D3 using the vitamin D2 peak at 907 cm-1 (Westermark, 1991). Typical FTIR spectra of crystalline vitamin D2 and D3 (Liu, Y., unpublished data) are shown in Figure 1.5. There is no published methodology on quantification of vitamin D using vibrational spectroscopy. The reason might be the inadequate detection limit and accuracy of the method since the concentration of vitamin D is normally low in foods. Vitamin D can be quantified by measuring its maximum UV absorption at 264 nm. The extinction coefficients for vitamin D2 and D3 are 19,400 and 18,300 M-1 cm-1 respectively. The limitation of this method is that other UV absorbing compounds in solution will interfere with the quantitation of vitamin D (Norman & Henry, 2007). 1.2.6.3  HPLC methods High-performance liquid chromatography (HPLC) methods have been widely  used for the determination of fat soluble vitamins since the mid-1970s and have been adopted as official methods due to good selectivity and detection ability (Ball, 2006). Table 1.5 lists the details of AOAC official methods for analysis of vitamin D in dairy products. The analytical procedure normally involves saponification, liquid extraction by organic solvents, concentration by evaporation, and purification by separating the vitamin from other interfering compounds and HPLC quantification. Reverse phase columns are dominant in the HPLC determination for vitamin D. After extensive sample preparation steps, the vitamin D extracts are injected into a reverse phase column and eluted by one or more solvents with or without gradient. Light exposure is avoided during the whole procedure.  28  Absorbance  Wavenumber (cm-1) Figure 1.5  FTIR spectra of crystalline vitamin D2 (grey line) and vitamin D3 (black  line). The differentiating peak of 907 cm-1 is marked by the arrow (Liu, Y., unpublished data).  29  Table 1.5  Summary of AOAC HPLC methods for vitamin D in dairy products.  Matrix  Saponification Solvent Purification Extraction  Column / Mobile phase  Detection  Reference  Fortified milk and milk powder  Aqueous KOH, 60 ºC, 30 min  Ethyl ether, pentane  Sil60D-10CN (250×4.6 mm)/ Amyl alcohol (0.35%) in hexane  Stainless Partisil (250×4.6 mm)/ Amyl alcohol (0.35%) in hexane  UV 265 nm  AOAC, 981.17 (Devries & Borsje, 1982)  Ready to feed milk based infant formula  KOH pellets, 75 ºC, 30 min  Ethyl ether, petroleum ether  Silica (250×4.6 mm)/ hexane-amyl alcohol (99.2+0.8)  Silica (150×4.6 mm)/ hexane- UV 254 amyl alcohol (99.85/0.15) nm  AOAC, 992.26 (Tanner et al., 1993)  Infant formulas and enteral products  Ethanolic KOH, 60 ºC, 30 min  Hexane  Silica SPE cartridge  C18 (250×4.6 mm)/ Gradient mixture of acetonitrile, methanol and ethyl acetate  UV 265 nm  AOAC, 995.05 (Sliva & Sanders, 1996)  Fortified milk, infant formula, margarine, milk powder  Aqueous KOH, 95 ºC, 30 min  n-Heptane  Silica (250×4.6 mm)/ 0.5% isopropanol2% Methy tertiary butyl ethercyclohexane–nheptane  C18 (250×4.6 mm)/ methanol-acetonitrile (20/80)  UV 265 nm  AOAC, 2002.05 (Staffas & Nyman, 2003)  30  Various temperature and time combinations have been reported for the saponification of food samples, ranging from room temperature, overnight to 95 °C, 30 minutes (Devries & Borsje, 1982; Tanner et al., 1993; Faulkner et al., 2000; Staffas & Nyman, 2003). Johnsson and Hessel (1987) reported that the ambient saponification was insufficient and might give uncontrollable emulsions. Official methods normally use the short time high temperature saponification to saponify fat content thoroughly. After the saponification, different solvents can be applied to extract vitamin D from aqueous phase of the samples. For example, hexane is normally used alone as the extracting solvent, but ethanol is sometimes added into hexane to prevent formation of an emulsion (Faulkner et al., 2000). Ethyl ether is often combined with petroleum ethyl or other solvents to extract vitamin D (Devries & Borsje, 1982; Tanner et al., 1993). During the saponification, vitamin D partially isomerizes into its precursor previtamin D (Johnsson and Hessel, 1987). In the four AOAC official methods for dairy products, various methods are used to correct the conversion from vitamin D to previtamin D. The AOAC 981.17 applies a correction factor of 1.25 on the measured vitamin D (Devries & Borsje, 1982) and AOAC 992.26 assumes 14% of the vitamin D converted into previtamin D during the saponification (Tanner et al., 1993). An internal standard of vitamin D2 is used in AOAC 995.05 and AOAC 2002.05 since the conversion rates of vitamin D2 and D3 to the respective previtamins are similar (Sliva & Kanders, 1996; Staffas & Nyman, 2003). Before being applied to the analytical column, an extensive purification of the crude extract is needed. As shown in previous Table 1.5, most AOAC official methods use a normal phase column to separate vitamin D from interfering compounds. Samples need to be individually purified using a silica column and then collected for the determination on an  31  analytical column. In comparison, AOAC 995.05 uses a silica solid phase extraction (SPE) cartridge to clean up the crude extract, which allows multiple samples to be processed using a vacuum manifold. SPE is also applied in some other methods to replace the normal phase column for purification (Mattila et al., 1992; Renken & Warthesen, 1993; Hagar et al., 1994; Faulkner et al., 2000). Direct extraction using a SPE cartridge for vitamin D in non-fortified sample was also reported (Blanco et al., 2000; Iwase, 2000; Heudi et al., 2004; Bartolucci et al., 2011). Table 1.6 summarizes various SPE protocols involved in vitamin D determination. Antioxidants are often added to protect vitamin D before HPLC analysis. Pyrogallol, ascorbic acid or sodium ascorbate are used during saponification since they are water soluble (Faulkner et al., 2000; Trenerry et al., 2011). Butylated hydroxytoluene (BHT) is often used to protect vitamin D in organic solvent (Devries & Borsje, 1982; Grace & Bernhard, 1984; Staffas & Nyman, 2003). With high reproducibility and relatively short protocol, the HPLC method has become dominant for routine analysis of vitamin D.  32  Table 1.6  Solid phase extraction techniques for vitamin D determination.  SPE cartridges  Purpose  Analytes  Conditioning/  Wash  Elute  Reference  Hexane  Hexane/isopropanol (99.5/0.5)  Mattila et al., 1992  Extraction Mega Bond Elut Silica SPE column  Extract purification  D in egg yolk  Hexane  Bond Elute SPE silica cartridge  Extract purification  D in milk  Hexane  Hexane  Hexane/chloroform (21.5/78.5)  Renken & Warthesen, 1993  Sep-Pak Florisil  Extract purification  Vitamin D in milk  Methanol (3ml) Isopropanol (4ml) Hexane (7ml)  Hexane  Isopropanol  Hagar et al., 1994  Mega Bond Elut C18  Direct extraction  Fat-soluble vitamins  Methanol (25ml) and MIlli-Q water  Methanol/water (10/90)  Methanol  Blanco et al., 2000  Extract-clean Si  Extract purification  Vitamin D  Hexane 1-2 volumes  Hexane/chloroform (22/78)  Methanol  Faulkner et al., 2000  Bond Elute C18  Direct extraction  Vitamin D2 as emulsified nutritional supplements  0.2 M K2HPO4 aqueous solution with 1 mM EDTA·2Na·2H2O  Deionized water then 10% aqueous methanol  Methanol  Iwase, 2000  Chromabond XTR®  Direct extraction  Vitamin A, D3 & E  -  -  Hexane  Heudi et al., 2004  Supelclean LC-NH2  Direct extraction  Vitamin D3 in cod liver supplement  Hexane  Hexane  Ethyl acetate  Bartolucci et al., 2011  Hexane/isopropanol (99.5/0.5)  33  Chapter 2: Hypotheses and research outline 2.1  Hypotheses and objectives As introduced in the literature review, the non-compliance of vitamin D  fortification of milk has been a recurring problem and little information is available regarding the current situation of milk fortification in British Columbia since the last report in 1993. The reasons behind the fortification problem are also not clear despite frequently reported non-compliance. In this situation, information about the stability of vitamin D during processing and storage may provide some hints for processors to fortify foods with targeted concentration. The purpose of the present research is to investigate whether the non-compliance of vitamin D fortification still exists in fluid milk sold in Vancouver and whether the instability of vitamin D might be a reason for the variation of vitamin D content in milk.  Hypothesis 1: Non-compliance of vitamin D fortification exists in fluid milk sold in Vancouver. To examine this hypothesis, vitamin D3 content of commercial fluid milk from seven brands in Vancouver grocery stores was analyzed.  Hypothesis 2: Different commercial formulations of vitamin D3 differ in their stability to light exposure during storage. To examine this hypothesis, four commercial formulations of vitamin D were added into skimmed milk and exposed to light at 2000 lux intensity for 22 days. Vitamin D retention of each formulation in milk was calculated and compared.  34  Hypothesis 3: Vitamin D2 and D3 have different stabilities at different pH values during thermal treatment. To examine this hypothesis, crystalline vitamin D2 and D3 content in pH 3.5 and 6.6 buffers were analyzed before and after thermal treatment of 72°C for 15 seconds.  35  Chapter 3: Vitamin D of fortified fluid milk in Vancouver Milk fortification with vitamin D is mandatory in Canada. However, wide variation has previously been noticed in vitamin D content of fortified milk (Chen et al., 1993; Faulkner et al., 2000). Moreover, the non-compliance of vitamin D fortification has been reported from time to time. Some recent studies indicated that the non-compliance of milk fortification is still a problem in the US (Murphy et al., 2001; Patterson et al., 2010) while the last available information about vitamin D in BC fluid milk was published in 1993 (Chen et al., 1993). This investigation was conducted to acquire the information on vitamin D of fluid milk in Vancouver. In total 104 cartons of fluid milk from seven brands were purchased from Vancouver grocery stores from November 2011 to September 2012. The vitamin D content in these products was analyzed on their "Best Before Date" or previous day and compared with the CFIA requirement (Figure 3.1). 3.1 3.1.1  Materials Chemicals and apparatus Potassium hydroxide, ascorbic acid, butylated hydroxytoluene, sodium sulphate,  hexane, and absolute ethanol were purchased from Fisher Scientific Canada (Ottawa, ON) and were of reagent grade or higher. Dichloromethane, methanol, isopropanol and ethyl acetate from Fisher Scientific were HPLC grade. Vitamin D2 (ergocalciferol, E5750), and vitamin D3 (cholecalciferol, C9756), were purchased from Sigma Aldrich Canada (Oakville, ON).  36  Milk of seven brands in Vancouver grocery stores  Skim milk  2% or 3.25% milk  2 - 3 replicates from  2 - 3 replicates from  each container  each container  ×4 Four sets of samples  Figure 3.1  Overview of sampling of commercial milk products for vitamin D  measurement.  37  Agilent 1100 Series LC (Mississauga, ON) with quaternary pump system and diode array detector was used for vitamin D determination. A Grace Vydac 201TP54 C18 column (Deerfield, IL) of 4.6 mm internal diameter and 250 mm length was installed on the HPLC system as the analytical column. A Vydac 201TP C18 guard column (Deerfield, IL) was installed to protect the analytical column. 3.1.2  Vitamin standards Vitamin D3 standard solutions were prepared in methanol at concentrations  ranging from 0.200 to 4.000 µg/mL (8-160 IU/100 mL). Vitamin D2 internal standard solutions were prepared in methanol at concentrations of 50.0 µg/mL and 5.00 µg/mL for calibration samples and milk samples respectively. The concentration of vitamin D3 standards and vitamin D2 internal standard were verified by UV absorption at 264 nm. All vitamin D3 standard solutions and vitamin D2 internal standards were stored in amber vials and kept at -80 °C when not in use. Solutions were discarded after two weeks and new standards were made. Calibration samples were prepared daily by adding 20 µL internal standard to 1 mL of vitamin D3 standard solution. 3.1.3  Sampling of commercially fortified fluid milks Fluid milk from seven brands was collected from retail stores on four separate  occasions (referred to as four "sets") from November 2011 to September 2012. The exception was brand 2, which was only collected in two sets as it was withdrawn from the Vancouver market and was no longer available after the second set was sampled. Each set was purchased after the procurement of the previous set was completed. In a few instances, sampling had to be repeated for a particular brand in a set due to analytical  38  problems, and thus occasionally there was an overlap in the time periods for some samples across the sets. For each set, two containers of each milk product (i.e. with the same lot number) were purchased from the same location. To evaluate the potential effect of milk fat content on fortification, skim milk and 3.25% milk were selected for the analysis, except for brand 6, for which 2% milk was used since 3.25% milk was not found. Table 3.1 shows an overview of the milk products. Samples of brand 1 were in 1 litre clear glass bottles, samples of brand 2 were packed in 1 litre opaque plastic containers, and the milk of the other five brands were in 1 litre laminated paperboard cartons. Brand 5 and Brand 7 were produced by the same dairy processor. The processor of brand 2 was unknown since this brand was withdrawn from the market after the  second set, thus the  information of processor was not available anymore. In other words, the seven brands represented fluid milks from at least 5 different dairy processors as identified by the CFIA Registered Dairy Establishment 4 digit codes. All milks were immediately stored in a cold room at 4 °C after purchase. Since the production date for each sample was not available on milk package, the vitamin D was measured either on the "Best Before Date" or the day before this date. Details of the sampling dates and locations as well as analysis dates for each of the commercially fortified fluid milks can be found in Appendix A. 3.2  Vitamin D determination Among different types of methods for vitamin D determination, the HPLC  methods have been dominant and have been accepted as AOAC official methods for measuring vitamin D in various food samples. Based on the equipment availability, AOAC method 995.05 was adapted for the vitamin D measurement in this research. 39  Table 3.1  Overview of milk samples of seven brands.  Brand  Processor  Milk type  Package  Volume  1  A  Skim & 3.25%  Clear glass bottle  1 litre  2  Unknown  Skim & 3.25%  Opaque plastic container  1 litre  3  B  Skim & 3.25%  Laminated paperboard cartons  1 litre  4  C  Skim & 3.25%  Laminated paperboard cartons  1 litre  5  D  Skim & 3.25%  Laminated paperboard cartons  1 litre  6  E  Skim & 2%  Laminated paperboard cartons  1 litre  7  D  Skim & 3.25%  Laminated paperboard cartons  1 litre  40  3.2.1  Saponification Samples were prepared according to AOAC method 995.05 (Sliva & Kanders,  1996) with slight modification. The milk container was inverted 20 times in case there was any stratification of vitamin D, then an aliquot of about 15 mL (15.20 g weighed accurately) of milk was added into a 125 mL Erlenmeyer flask. To the flask was added 20 µL vitamin D2 internal standard, 1.5 mL of ascorbic acid solution in water (19.4% wt/vol), and 19 mL potassium hydroxide solution (30.7% wt/vol in 20:163 ethanol:water vol/vol). The flask was stoppered and heated in a shaking water bath (Blue M, Blue Island, Illionois) at 60 °C for 30 min. Aluminum foil was used to cover the water bath to avoid light exposure. 3.2.2  Extraction The sample mixture was transferred to a 250 mL clear separatory funnel along  with 15 mL water and 60 mL hexane was used to rinse the flask. The contents were shaken for 90 s and the layers were allowed to separate. The aqueous layer was drained and the organic phase washed with 15 mL water. The aqueous layer was drained to waste and the sample mixture washed again with 15 mL of water after being adjusted to neutrality by drop wise addition of 10% acetic acid (vol/vol) using phenolphthalein as indicator. The aqueous layer was drained. The organic phase was passed through sodium sulfate into a 100 mL round bottom flask and to it was added 1 mL of BHT dissolved in hexane (1 mg/mL). The solvent was removed under vacuum using a rotary evaporator (Buchi, Flawil, Switzerland) with a water bath temperature of 40 °C.  41  3.2.3  Sample purification The crude extract was purified on a vacuum manifold (Honeywell Burdick  Jackson, Morristown, New Jersey, USA) using Sampliq silica SPE cartridges (3 mL capacity, 500 mg packing, Agilent Canada) that had been conditioned with 4 mL dichloromethane-isopropanol solution (80:20 vol/vol) and then 5 mL dichloromethaneisopropanol solution (99.8:0.2 vol/vol). The crude sample extract was dissolved in 2 mL dichloromethane-isopropanol (99.8:0.2 vol/vol) and transferred to the cartridge along with a 1 mL rinse of dichloromethane-isopropanol (99.8:0.2 vol/vol). The cartridge was then washed with 2 mL dichloromethane-isopropanol (99.8:0.2 vol/vol). Finally, the sample was eluted with 7 mL dichloromethane-isopropanol (99.8:0.2 vol/vol) into a clean test tube. The purified sample was filtered through Millex 0.45 µm 4 mm PTFE syringe filter (Millipore, Billerica, Massachusetts) and then evaporated in a Rapidvap Vertex Evaporator (Labconco, Kansas City, Missouri). The dried sample was transferred to an HPLC vial and then dried under a stream of nitrogen. The residue was reconstituted in 100 µL methanol/dichloromethane (80:20 vol/vol) for analysis by HPLC. 3.2.4  HPLC analysis of vitamin D The injection volume was 40 μl out of the 100 μl concentrated sample. Vitamin  D2 and D3 were eluted by isocratic mobile phase of 98% acetonitrile and 2% methanol. Then the column was washed using 98% ethyl acetate and 2% methanol. By the end of the run, the mobile phase was switched back to acetonitrile and methanol. The total running time was 26 minutes. The mobile phase change is shown in Table 3.2. Vitamin D3 and the internal vitamin D2 standard were monitored at 264 nm. A six-point calibration curve was constructed before the sample analysis on a daily basis. 42  Table 3.2  Flow rates and concentrations of mobile phase components for determination  of vitamin D in fluid milk by HPLC. Time, min  Flow rate, mL/min  Acetonitrile, % Methanol, %  Ethyl acetate, %  0.0  1.2  98  2  0  15.5  1.5  98  2  0  17.0  1.5  0  2  98  20.0  1.2  0  2  98  21.5  1.2  98  2  0  43  The area ratio of the vitamin D3 standard peak and the vitamin D2 internal standard peak were plotted against vitamin D3 standard concentration. Vitamin D concentration in the sample expressed as IU/100 mL was quantified by applying the vitamin D3:vitamin D2 ratio of the sample on the equation derived from the six-point standard calibration curve. 3.3  Statistical analysis Three-way ANOVA and Tukey's test (Minitab 16, State College, PA) was  conducted to determine the factors contributing to significant variation of vitamin D (p<0.05). Brand, milk fat content and sampling sets were defined as potential factors for vitamin D variation. 3.4 3.4.1  Results and discussion Establishment of the adapted AOAC official method Currently there are four AOAC official methods for vitamin D measurement in  dairy products. The AOAC 995.05 method was selected and adapted for this research because it uses SPE cartridge to purify the vitamin D extract. Comparing with LC column purification, SPE speeds up the preparation for each sample while removing most interfering compounds. Furthermore, multiple samples can be purified and concentrated simultaneously. In many AOAC official methods measuring vitamin D, ascorbic acid or sodium ascorbic acid were used in saponification and BHT during evaporation on water bath (Devries & Borsje, 1982; Tanner et al., 1993; Staffas & Nyman, 2003). In the original 995.05 method, none of these anti-oxidants are applied. To protect vitamin D3 and internal standard of vitamin D2 in the present study, ascorbic acid was added together  44  with the internal standard before saponification in water bath and BHT solution was added before rotary evaporation at 60 °C. In the original AOAC 995.05 method, the HPLC running time is 35 minutes with gradient mobile phase. The flow rate was increased to speed up the HPLC analysis in this research. System suitability is to verify that the whole system of instrument, reagent, column and samples are suitable for the intended method. Vitamin D2 and vitamin D3 solutions were stored at room temperature for four days to cause degradation of vitamin D to previtamin D. The system suitability was tested using the stressed vitamin D2 and vitamin D3 solutions. The resolution of vitamin D peaks and the reproducibility were examined. Figure 3.2 shows that the peaks of previtamin D and vitamin D were well separated. In the original method, the ratio of vitamin D2 : vitamin D3 is calculated using peak height. In this research, the peak area ratio was applied instead of peak height to improve the precision of quantification. The test of replicate injections showed that relative standard deviation (RSD) of vitamin D3 calculation from 5 injections ranged from 0.7% to 5.5% for concentrations ranging from 9 to 0.3 µg/ mL. Recovery was calculated by spiking fortified milk in three replicates. Vitamin D was added to the skim and 3.25% milk at 3.6 and 36 IU/100 mL. The low concentration spiking showed a recovery of 96% and 102% for skim and 3.25% milk respectively. The high concentration spiking was recovered at 100% and 101% for skim and 3.25% milk respectively. Baseline drift was observed during method verification, thus a reference wavelength of 345 nm was used to correct for any baseline drift. 45  mAU b d 14 12 10 8 6 4 2 0  a 0  Figure 3.2  2  4  6  8  10  c 12  c 14  16  18  HPLC chromatogram of vitamin D2 and vitamin D3 standards.  Peak identification: a) previtamin D2; b) vitamin D2; c) previtamin D3; d) vitamin D3.  46  The criteria of acceptable HPLC performance were as follows. Shift of retention times should not be more than ± 0.3 min. Peaks should be nearly symmetrical with only minimal tailing at high concentrations. System pressure should not exceed 100 bar. Standard curves should have a minimum of five points and correlation factor r2 no less than 0.997. Figure 3.3 is an example of the standard curve. The column was cleaned thoroughly when contamination was indicated by peak shift. The column was reversed in order to flush out contaminants accumulated at the front part of the column. Four solvents were used in this procedure at a flow rate of 1 mL/min. The change of mobile phase is shown in Figure 3.4. Each solvent was run at 100% for 15 min before switching to the next solvent. After the wash from acetonitrile to ethyl acetate, the solvent was switched back from 100% ethyl acetate back to 100% acetonitrile. Each solvent was run about 5 min in this step. Guard column replacement was also conducted to improve the performance of analytical column after thorough cleaning. 3.4.2  Vitamin D in commercially fortified fluid milk Two containers of the same product were purchased in each of the four sets in  case there was container to container variation in vitamin D content. The measured vitamin D content for each container ("A" and "B") of the same product in each set and the results of paired comparison of vitamin D in the two containers are shown in Appendix B. Student t-test analysis showed that there was no significant difference (p>0.05) between container A and B with the exception of the two containers of 3.25% milk for brand 5 in set 2 (p=0.0493). Therefore, in this research the total four or six replicate data measured from both containers of the same product in each set were considered as one data for subsequent data analysis. 47  5  Area Ratio  4  y = 1.1064x - 0.0222 R²= 0.9998  3 2 1 0 0  1  2  3  4  Vitamin D3 (ug/mL) Figure 3.3  Standard curve of vitamin D3 determination using HPLC (vitamin D2 peak  area ratio versus vitamin D3 concentration).  48  Acetonitrile  Methanol  Acetonitrile  Figure 3.4  Isopropanol  Methanol  Ethyl acetate  Isopropanol  Sequence of solvents used for washing and re-conditioning the analytical  HPLC column.  49  The mean vitamin D content and standard deviation of each product in each set are shown in Table 3.3. All milk samples were labelled as containing 45% of the daily value of vitamin D3 per 250 mL, which represents 36 IU/100 mL based on 200 IU as the recommended daily allowance according to the Food and Drug Regulations (2012), since the new recommended daily value of 600 IU has not yet been adopted in the regulations. Generally, fortification of 3.25% or 2% milk was more compliant than that of skim milk. CFIA (2012) requires that the vitamin content in fortified milk should be within the range of 35.2 to 46.9 IU/100 mL and the processors should target the final concentration of 41 IU/100 mL. Only 42% of the 104 cartons of sampled milks in this study were compliant with this requirement. The compliance of milk samples with higher fat content was better than that of skim milk, with 57% of 2 or 3.25% milk and 27% of skim milk satisfying the CFIA standard respectively. Under-fortification was the major problem found in milk samples in this research, especially skim milk. In total 73% of skim milk samples and 35% of 2 or 3.25% milk samples were found to be under-fortified. None of the skim milk samples contained vitamin D greater than 46.9 IU/100 mL, while 8% of milk with higher fat content were over-fortified. As demonstrated in Figure 3.5, in general higher vitamin D3 content was found in 2 or 3.25% fat content milk than skim milk. Three-way ANOVA confirmed that significant difference existed among different brands, sampling sets, and products with different fat content. In addition, the interaction between brand and milk fat content was also significant. The results are summarized in Table 3.4. The vitamin D content in skim milk of brands 5 and 7 were consistently low, with the mean value of four sets ranging from 8.53 to 15.1 IU/100 mL (Table 3.3), which were 50  Table 3.3  Vitamin D3 content (mean ± standard deviation, n = 4 or 6) of seven brands  of fluid milks collected in four sampling sets. Milk  Vitamin D3 content (IU/100mL)  fat Brand 1 Skim 3.25% Brand 2 Skim 3.25% Brand 3 Skim 3.25% Brand 4 Skim 3.25% Brand 5 Skim 3.25% Brand 6 Skim 2% Brand 7 Skim 3.25% a  Set 1  Set 2  Set 3  Set 4  32.2 ± 1.3  36.5 ± 1.0  1.3 ± 0.1  35.0 ± 0.6  29.5 ± 1.4  29.4 ± 0.3  25.8 ± 0.7  26.8 ± 0.3  23.0 ± 0.7  31.0 ± 0.6  N/Aa ± N/A  N/A ± N/A  39.4 ± 1.1  35.9 ± 0.9  N/A ± N/A  N/A ± N/A  45.6 ± 0.6  33.3 ± 1.1  36.8 ± 0.8  31.3 ± 0.8  43.2 ± 0.9  42.9 ± 0.9  27.5 ± 2.0  30.3 ± 1.1  34.5 ± 0.4  31.2 ± 1.1  40.8 ± 0.5  44.5 ± 0.9  40.9 ± 0.9  41.8 ± 0.7  35.5 ± 1.6  57.3 ± 0.4  15.1 ± 0.7  14.1 ± 0.5  9.9 ± 0.4  13.7 ± 1.6  32.2 ± 0.9  47.3 ± 0.8  36.6 ± 1.5  41.3 ± 0.4  32.8 ± 1.1  38.8 ± 0.8  34.3 ± 0.8  39.3 ± 1.0  38.7 ± 1.6  33.9 ± 0.6  35.6 ± 0.2  40.5 ± 1.1  12.0 ± 0.7  11.7 ± 0.3  8.5 ± 0.3  14.3 ± 0.2  45.1 ± 0.8  13.7 ± 0.9  35.2 ± 1.1  38.0 ± 0.9  N/A = data not available  51  2% or 3.25% milk *  Skim milk  60  IU/100 mL  50 40 30 20  10 0 Brand 1 Figure 3.5  Brand 2  Brand 3  Brand 4  Brand 5  Brand 6  Brand 7  Comparison of vitamin D3 content in skim milk versus milk with 2 or 3.25%  fat. Values shown are mean and standard deviation of four sets (n≥8). (* 2% fat milk was analyzed for brand 6; 3.25% fat milk was analyzed for other brands)  52  Table 3.4  Results of three way ANOVA for vitamin D in commercial fluid milk.  Factors  DF  Seq SS  Adj SS  Adj MS  F value  Probability  Brand  6  9325  9364  1561  38.8  0.000  Milk fat content  1  4614  4745  4745  117.9  0.000  Sampling set  3  1467  1371  457  11.4  0.000  Brand ×Milk fat content  6  5742  5742  957  23.8  0.000  Set ×Milk fat content  3  171  164  55  1.4  0.258  Error  218  8776  8776  40  Total  237  237  30096  53  less than 50% of the claimed amount. Brands 5 and 7 are produced by the same milk processor according to the registration number marked on the package. Compared to skim milk, milk with 3.25% fat of these two brands showed better fortification compliance with vitamin D content close to the claimed value. Wide variation was also observed in the vitamin content in milk products with same fat content but from different brands or sampling sets. For example, only trace amount of vitamin D3 content was found in set 3 skim milk of brand 1, while the first two sets and the fourth set contained vitamin D3 content close to each other (Table 3.3). A dramatically lower vitamin D content was also observed in 3.25% milk of set 2 of brand 7. The inconsistency in vitamin D3 content in the same products from different sets can be visually shown by the standard deviation of vitamin D content of the same product in four sets, which was demonstrated as error bars in Figure 3.5. The product with longer error bars means big variation was found among the samples in different sets. Except for brand 6, fluctuating vitamin D contents were observed in the other brands, either in skim milk or 3.25% milk, or both products. Brands 1 and 2 were packed in glass bottles and plastic jugs respectively, while paperboard cartons were used in the other five brands. As a result, vitamin D loss might be greater in brands 1 and 2 since vitamin D is light sensitive. As expected, the mean vitamin D content in 3.25% milk of brand 1 was the lowest among the seven brands. However, its skim milk had a moderate vitamin D concentration comparing with other brands. Both skim milk and 3.25% milk of brand 2 contained vitamin D at a moderate level among the seven brands. The impact of different container on vitamin D content was not very clear.  54  3.5  Conclusions Wide variation among brands or sets in vitamin D content was found in milk  samples purchased in Vancouver. The fortification non-compliance was observed in 58% of the products sampled. There are several possible reasons contributing to the variation and non-compliance of vitamin D fortification in milk products. Commercial vitamin D3 products used for fortification may have different stability during storage. Different fortification protocol might be applied by milk producers. The fortification of skim milk is more difficult than 2% or 3.25% milk since vitamin D is fat soluble. All of these factors may cause variation in vitamin D content in final products. More information about the fortification practices of milk producers indicating the type and stability of vitamin D formulations used will be helpful to improve the compliance of vitamin D fortification in milk.  55  Chapter 4: Vitamin D in laboratory fortified milk exposed to light Vitamin D is sensitive to light. Published studies confirmed that light is a significant factor contributing to vitamin D loss in fortified milk (Renken & Warthesen, 1993). Riboflavin, as a photo-sensitizer, produces singlet oxygen on light exposure and thus trigger the oxidation of vitamin D in milk (King & Min, 1998). Since riboflavin is naturally present in milk, significant loss of vitamin D may occur on light exposure when milk is packed in clear plastic bags or glass bottles. Milk in plastic jugs with various light transmittance might be impacted as well (Saffert et al., 2008 & 2009). According to the Food and Drug Regulations (2012), vitamin A and D fortification is mandatory for skim milk while in other fluid milk products, only vitamin D addition is required. Vitamin A is sensitive to light exposure and the added vitamin A might be even more sensitive than indigenous vitamin A in milk (Gaylord et al., 1986; Bartholomew & Ogden, 1990). The impact of vitamin A degradation on vitamin D is still unknown. Commercial vitamin D formulations may differ in the stability to light exposure because of the different encapsulating materials or emulsifiers. In this research, four commercial vitamin D formulations were used to fortify milk and the vitamin D retention on light exposure was measured during three weeks of storage at 4 °C. In-house fortified skimmed milk samples were used to investigate the impact of light exposure on four different commercial formulations of vitamin D3 during cold storage (Figure 4.1). The vitamin D content during the storage of light-exposed samples was compared with that of the corresponding control samples stored in the dark.  56  Pasteurized skimmed milk  Crystalline D3 + VA*  SDS D3 + VA  CWS D3 + VA  AD premix  Three bottles for each fortified milk  Two bottles exposed to light  One bottle stored in the  for each formulation  dark for each formulation  2 - 3 replicates tested at day 1, 8, 15 and 22 for each bottle  Figure 4.1  Overview of study design to test stability of different vitamin D  formulations in milk during storage with light exposure. * VA = vitamin A  57  4.1 4.1.1  Materials Commercial Vitamin D Formulations Vitamin D formulations were donated by DSM Nutritional Products (Belvidere,  New Jersey, USA) and Kingsway Chocolate (Mississauga, Ontario, Canada). Vitamin A, in the form of a water dispersible powder of retinyl palmitate, was donated by DSM Nutritional Products. Two of the vitamin D formulations from DSM Nutritional Products came in the form of water dispersible encapsulated powders, dry vitamin D3 100 CWS/AM (CWS D3) and dry vitamin D3 100 SD/S (SDS D3), while the third vitamin D used in this research was food grade crystalline vitamin D3 (crystalline D3).  The  formulation from Kingsway Chocolate came in the form of an emulsified mixture of vitamin D3 and vitamin A palmitate (AD premix). The ingredients of the vitamin formulations are summarized in Table 4.1. The detailed product information of each formulation is attached as Appendix C. Erythromycin (E0774) was purchased from Sigma-Aldrich (Oakville, ON). 4.1.2  Preparation of fortified milks for the study of stability of vitamin D to light  exposure Raw whole milk donated by the UBC Dairy Education and Research Centre (Agassiz, BC) was skimmed by centrifugation at 10,000 g at ambient temperature for 20 minutes on a Sorvall RC 5B Plus centrifuge (Thermo Fisher Scientific, Ottawa, ON). The skimmed milk was stored immediately at -30 °C before thermal treatment. The skimmed milk was thawed under cold running water, then pasteurized at 72 °C for 15 seconds using a FT74X HTST/UHT Heat Exchanger Processing Unit at the Food Technology  58  Table 4.1  Ingredients of vitamin formulations used in the present research.  Vitamin formulations  Ingredients  Crystalline D3  Vitamin D3  SDS D3  Vitamin D3, medium chain triglycerides, modified food starch, sucrose, dl-α-tocopherol, sodium ascorbate and silicon dioxide  CWS D3  Vitamin D3, medium chain triglycerides, corn starch, acacia gum and sucrose, dl-α-tocopherol, and silicon dioxide  AD Premix *  Vitamin D3, vitamin A palmitate, sunflower oil, polysorbate 80, polyglycerol monooleate  Water dispersible  Vitamin A palmitate, corn starch, gelatin, sucrose and dl-α-  vitamin A  tocopherol.  * The vitamin supplier suggested that the AD premix containing 10 mg of mixed tocopherol and 5 mg of BHT per 1.7 million IU of vitamin A palmitate although such information was not included in Appendix C.  59  pilot plant of British Columbia Institute of Technology (BCIT) (Burnaby, BC). Pasteurized milk was stored at -30 °C before fortification. Stock solutions of CWS D3 and SDS D3 were prepared by dissolving in water. Ethanol was used for preparing the crystalline D3 stock solution, while vitamin A and D premix was dissolved in 75% ethanol and 25% dichloromethane. All vitamin D3 stock solutions were made to target concentration of 320 IU/mL. For the purpose of comparison to the emulsified vitamin AD premix from Kingsway Chocolate, vitamin A dissolved in water to 6663.8 IU/mL was used as the stock solution for addition to the three milk samples fortified with CWS D3, SDS D3 and crystalline D3. Milk samples were fortified with the prepared vitamin stock solutions to a final vitamin D concentration of 40 IU/100 mL and to a final vitamin A concentration of 218 IU/100 mL by adding a 2 mL aliquot of vitamin stock solution into 1.6 L of milk. Erythromycin was added at 100 mg/L at the same time of fortification to minimize the growth of microorganisms during the three weeks of storage. Milk was stirred for 10 minutes after the addition of vitamins and erythromycin. Each fortified milk sample was portioned into three 500 mL glass bottles (Avalon Dairy, Burnaby, BC), with two bottles exposed to light as described in Figure 4.2, and one control bottle which was wrapped in aluminum foil to prevent light exposure. 4.1.3  Chemicals and apparatus for vitamin D and vitamin A determination The chemicals and apparatus for vitamin D were as described in section 3.1.1.  Vitamin A in the form of retinyl palmitate (R3375) was purchased from Sigma-Aldrich (Oakville, ON). Retinyl propionate (sc-236667) was purchased from Santa Cruz  60  Crystalline D3  2,000 Lux  AD premix  O  C  SDS D3  C  Lamp 2,000 Lux  CWS D3  O Lamp  O  C  C  O  C  C  O  O Lamp  SDS D3  CWS D3  Crystalline D3  Lamp  AD premix  (a)  (b)  (c) Figure 4.2  Set up for the exposure of fortified skimmed milk samples to light.  (a) Schematic of top view: skimmed milk fortified with four vitamin D formulations were aligned along the line of 2,000 lux, which was typical light intensity of showcase in local grocery. C - center position; O - outside position; (b) Schematic of front view; (c) photograph of light exposed samples. 61  Biotechnology (Santa Cruz, CA) and served as the internal standard for vitamin A determination. Ethanol, methanol, hexane, isopropanol, acetonitrile, and ethyl acetate were purchased from Fisher Scientific Canada (Ottawa, ON) in HPLC grade. Vitamin A stock solutions ranging from 0.20 to 3.96 μg/mL and internal standard of 10 μg/mL were prepared in isopropanol and then in methanol by serial dilution. The vitamin A standards were prepared by mixing 100 μL internal standard, 400 µL vitamin A stock solution, and 500 µL dichloromethane in a HPLC vial. 4.2  Experimental set-up for the light exposure study Milk samples were exposed to 2000 lux light intensity over the course of 22 days  storage at 4 °C. Two fluorescent lamps (Blue Planet 052-5109-0 fluorescent tubes with colour temperature of 4,100 K; Canadian Tire, Vancouver, BC) were used as the source of light. As shown in Figure 4.2, positions on both side of the lamps providing 2000 lux intensity were determined by measuring light intensity using a LuxMaster light meter (Harry’s Pro Shop, Toronto, ON). For each vitamin D formulation, the control milk was stored in the dark at 4 °C, the other two bottles were placed along the 2000 lux lines, at center and outside positions respectively. 4.3  Analysis of vitamin D and vitamin A Milk was analyzed for vitamin D content in triplicate from light exposed bottles  on day 1, day 8, day 15, and day 22 of the fortification. The control bottle was analyzed in duplicate at the same time. Vitamin D was determined using the HPLC method described in section 3.2. The retention of vitamin D in each fortified milk was calculated relative to the vitamin D content of the corresponding control milk stored in the absence of light. 62  Milk samples were extracted for vitamin A, retinyl palmitate based on the method of Hite (2003). In a 16×100 test tube, 0.25 mL skim milk, 0.25 mL water and 1.25 mL ethanol were added with 10 μL internal standard, then mixed on vortex for 30 seconds. The mixture was allowed to stand still for 5 min before adding 1.25 mL hexane, then vortexed again for 30 seconds. After 2 min, the addition of hexane and vortex was repeated two more times, then 0.75 mL water was added and the sample was mixed on vortex for 5 seconds. The mixture was centrifuged at 633 g for 10 min. The top hexane layer was filtered through a 0.45 μm syringe filter, then evaporated under nitrogen flush and reconstituted in 100 μL 50:50 CH2Cl2/MeOH for HPLC analysis. Vitamin A was analyzed at 325 nm on an HPLC system using the same columns described in section 3.1.1 at the column temperature of 24 °C. A gradient mobile phase of 95:5 acetonitrile/water to 100% methanol was applied, followed by ethyl acetate wash (Table 4.2). The total running time was 34 min. 4.4  Microbial total plate count 3M Petrifilm Aerobic Count Plates (3M Canada, London, ON) were used to  evaluate the total microbial counts. At the end of the storage of the milk samples, samples were diluted aseptically from 10-1 to 10-6 using peptone water (0.1% wt/vol) and incubated at 32 °C for 48 hours in an Innova®40 incubator (New Brunswick Scientific, Enfield, CT, USA) before conducting the microbial counts. Microbial analysis was conducted in duplicate.  63  Table 4.2  Flow rates and concentrations of mobile phase components for determination  of vitamin A in fluid milk by HPLC. Time,  Flow rate,  min  mL/min  Acetonitrile, %  Methanol, %  Ethyl acetate, %  Water, %  0.0  1.5  95  0  0  5  3.5  1.5  95  0  0  5  8.0  1.5  0  100  0  0  19.0  1.5  0  100  0  0  22.0  1.5  0  5  95  0  27.0  1.5  0  5  95  0  30.0  1.5  95  0  0  5  64  4.5  Statistical analysis Statistical analysis was performed using Minitab 16 (State College, PA). Three-  way ANOVA was conducted using general linear model analysis for repeated measures. Tukey's test was applied to investigate the significant variation in vitamin D content among the different formulations and changes with time. Regression analysis was conducted to fit the degradation of vitamin D over time. 4.6  Results and discussion Vitamin D contents measured at days 1, 8, 15 and 22 for milk samples fortified  with different vitamin formulations are shown in Table 4.3. Vitamin D3 content dropped dramatically in all milk samples after being exposed to 2000 lux light for 22 days, while the control group retained vitamin D3 at a similar level to that measured at day 1. The aerobic microbial count in milk samples ranged from 0 to 108 CFU/mL, but no correlation was found between the vitamin D retention and microbial counts (r = 0.147, n=2). The results of three-way ANOVA indicated that the retention of vitamin D content was significantly different between milk samples fortified with the four vitamin D3 formulations (Table 4.4). Generally, vitamin D formulation, and exposure time were significant factors contributing to the variation of vitamin retention while no significant difference was observed between milk being stored in outside bottle versus center bottle. The interaction between sample position and vitamin D formulation was significant, indicating that vitamin D content of samples at different positions did not follow the same pattern in all formulations. The interaction of vitamin D formulation and exposure time  65  Table 4.3  Vitamin D3 content (mean and standard deviation or range*) during 22 days  of storage at 4 °C of milks fortified with four different vitamin formulations. Vitamin D3 content (IU/100 mL) Day  Crystalline D3  SDS D3  Outside  1  40.4 ± 0.6  37.4 ± 1.0  37.2 ± 0.7  40.5 ± 2.2  bottle  8  24.8 ± 0.9  26.4 ± 0.9  34.6 ± 0.2  25.4 ± 0.3  15  19.0 ± 0.6  18.3 ± 0.5  29.0 ± 0.5  15.5 ± 0.1  22  14.5 ± 0.4  13.8 ± 0.7  24.4 ± 0.9  10.6 ± 0.2  Center  1  40.4 ± 0.6  37.4 ± 1.0  37.2 ± 0.7  40.5 ± 2.2  bottle  8  21.9 ± 0.1  29.4 ± 0.3  34.1 ± 0.5  25.0 ± 1.1  15  17.6 ± 0.4  18.8 ± 0.0  28.6 ± 0.2  15.2 ± 0.1  22  14.7 ± 0.4  14.2 ± 0.7  23.8 ± 0.6  11.4 ± 0.5  1  40.4 ± 0.6  37.4 ± 1.0  37.2 ± 0.7  40.5 ± 2.2  8  37.9 ± 1.5  42.3 ± 2.5  38.1 ± 1.5  38.5 ± 0.4  15  37.6 ± 0.4  41.1 ± 0.9  38.2 ± 0.9  38.2 ± 1.1  22  37.7 ± 0.8  36.4 ± 0.9  38.0 ± 1.7  37.6 ± 0.5  Control  CWS D3  AD premix  * Standard deviation was calculated from the triplicate values of the outside and center bottles exposed to light, while range was calculated from the duplicate values of the control bottle held in the dark.  66  Table 4.4  Results of three-way ANOVA for retention of vitamin D3 in fortified  skimmed milk exposed to light. Factors  DF  Seq SS  Adj SS  Adj MS  F value  Probability  Vitamin D formulation  3  0.824  0.824  0.275  1830  0.000  Exposure time  3  4.611  4.611  1.537  10230  0.000  Sample position  1  0.000  0.000  0.000  1.74  0.191  Sample position ×  3  0.008  0.008  0.003  17.7  0.000  9  0.316  0.316  0.035  233  0.000  3  0.001  0.001  0.000  1.96  0.129  9  0.011  0.011  0.001  8.41  0.000  Error  64  0.010  0.010  0.000  Total  95  5.780  vitamin D formulation Vitamin D formulation × exposure time Sample position × exposure time Three-way interaction  67  was also significant, indicating that the vitamin D in different formulations degraded at different rates during the storage. The three way interaction was also significant. Each significant factor or interaction is further discussed in the following paragraphs. The trend of vitamin D retention in the four formulations can be seen in Figure 4.3. CWS D3 was obviously more stable than the other three vitamin D formulations. Since the mechanism of vitamin D degradation is not established, regression analysis using different models was tried to fit the degradation trend. The polynomial models gave the highest R2 values. Based on the four time points of data, the decrease in retention of SDS D3 followed a second order polynomial trend while the retention of CWS D3, crystalline D3 and AD premix was best fit using third order polynomial equations. The result of Tukey's test for mean vitamin retention at different storage time is shown in Table 4.5. It confirmed that the retention of vitamin D in CWS D3 samples was significantly greater than that in the other three groups after exposure to light. At day 8, SDS D3, AD premix and crystalline D3 had a similar retention of vitamin D on light exposure, while at day 15, retention of vitamin D in all formulations were significantly different from one another. In particular, the AD premix started to show greater vitamin D loss compared with the other three. At day 22, AD premix showed lowest retention of vitamin D while vitamin D in crystalline D3 fortified milk remained at a similar level to that of SDS D3 fortified milk. The crystalline D3 was expected to be labile to light since it was reported that vitamin D is sensitive to light especially when riboflavin is present (King & Min, 1998). The AD premix was an emulsified form and showed a lower stability than the crystalline  68  100% 90%  Retention of vitamin D  80% 70% 60% 50% 40% 30% 20% 0  5  10  15  20  25  Storage time (day)  Figure 4.3  Trends of percent retention of vitamin D3 content in skimmed milk fortified  with four different formulations during 22-day storage at 4 °C with light exposure.  × Crystalline D3   SDS D3  ▲  CWS D3  ○  AD premix  Crystalline D3  y = -0.0001x3 + 0.0051x2 - 0.0932x + 1.0882  R²= 0.990  SDS D3  y = 0.0014x2 - 0.0616x + 1.0607  R²= 0.992  CWS D3  y = 4E-05x3 - 0.0015x2 - 0.0034x + 1.0049  R²= 0.993  AD premix  y = 2E-05x3 + 0.0004x2 - 0.0544x + 1.054  R²= 0.998  69  Table 4.5 Tukey's test of vitamin D retention of four formulations with different light exposure times. Exposure time, days  Vitamin D retention, % Crystalline D3  SDS D3  CWS D3  AD premix  1  100 AW*  100 AW  100 AW  100 AW  8  62 BX  66 BX  90 AX  65 BX  15  49 BY  45 CY  75 AY  40 DY  22  39 BZ  38 BZ  63 AZ  29 CZ  * A, B, C, & D indicate significant difference between formulations (within row) and W, X, Y, & Z indicate significant differences between days of storage (within column).  70  D3. The significant difference between the retention of AD premix and crystalline D3 may be due to the instability of other compounds in the premix. The two encapsulated vitamin D3 forms showed different vitamin D retention. The CWS D3 was formulated by dissolving vitamin D3 in droplets of medium chain triglycerides, then encapsulated in the matrix of acacia gum and sucrose with a coating of corn starch. In SDS formulation, vitamin D3 was finely dispersed in a matrix of modified starch, sucrose and medium chain triglycerides. The properties and structure of encapsulation matrix may cause the variation of vitamin D stability to light. Despite having more surface area exposed to light, vitamin D in outside bottles was not always higher than center bottles (Figure 4.4). Furthermore, vitamin D content was not significantly different in milk placed at center and outside positions according to ANOVA analysis. A significant decline in vitamin D content was observed at each test day during the storage. This indicated the degradation of vitamin D in all formulations was ongoing during the 22-day storage. The interaction of sample position and vitamin formulation was significant, which means higher vitamin D retention was not always found in one position in all formulations. For example, as shown in Figure 4.4, crystalline D3 fortified milk in outside bottles retained more vitamin D than milk in center bottles at the 8th day. Milk fortified with SDS D3 showed a reversed pattern, in which more vitamin D was found in center bottles. Interaction of formulation and exposure time was also significant, which indicated that vitamin D in different formulations degraded at different rate, and this rate was changing during the storage, as shown by the trend lines in Figure 4.3. 71  100%  Crystalline D3 - O  80%  Crystalline D3 - C  60% 40% 20% 0% Day 1  Day 8  100%  Day 15 SDS D3 - O  Day 22 SDS D3 - C  80% 60% 40%  Vitamin D retention  20% 0% Day 1  Day 8  100%  Day 15  Day 22  CWS D3 - O  CWS D3 - C  Day 15  Day 22  80% 60% 40% 20% 0% Day 1  Day 8  100%  AD premix - O  AD premix - C  80% 60% 40% 20% 0% Day 1 Figure 4.4  Day 8  Day 15  Day 22  Vitamin D % retention in outside bottle (- O) and center bottle (- C) of  skimmed milk fortified with four vitamin D3 formulations (n=3). 72  Vitamin D retention  Day 8 100% 90%  Crystalline D3  80%  SDS D3  70%  CWS D3  60%  AD premix  50% Outside bottle  Center bottle  Vitamin D retention  Day 15 90% 80% 70% 60% 50% 40% 30%  Crytalline D3 SDS D3 CWS D3 AD premix Outside bottle  Center bottle  Vitamin D retention  Day 22 70% 60% Crystalline D3  50%  SDS D3  40%  CWS D3  30%  AD premix  20% Outside bottle  Figure 4.5  Center bottle  Vitamin D retention as a function of the interaction of formulation and  position at different exposure times.  73  The two way interaction between vitamin D formulation and sample position or exposure time were both significant. The significant three way interaction indicated the interaction between two factors changes when introducing the third factor. For example, the interaction of formulation and position was changed by the exposure time. The interaction of formulation, position and exposure time was plotted in Figure 4.5. The interaction between the formulation and time exposure also followed a different pattern in the outside bottle and center bottle, as shown in Figure 4.6. Since vitamin A and D fortification is mandatory for skim milk (Food and Drug regulations, 2012), industry usually uses a premix of vitamin A and D for skim milk fortification. For the purpose of comparison with vitamin AD premix, water dispersible vitamin A was added into the milk samples fortified with the other three vitamin D formulations, and the contents of vitamin A were analyzed for the samples collected at day 8 and 22 of storage at 4 °C. The results are summarized in Table 4.6. Vitamin A in milk samples were almost degraded completely on light exposure by the end of the storage. However, in the control bottles kept in the dark, more than 90% of vitamin A still remained in milk fortified with AD premix while little water dispersible vitamin A remained in control milk fortified with the other three vitamin D formulations. A followup experiment was conducted, in which skimmed milk was fortified with only the water dispersible vitamin A or together with either CWS D3 or crystalline D3, and stored in the dark at 4 °C. The results showed that the degradation of water dispersible vitamin A started within one week even in the dark (Table 4.7). The vitamin D loss in this research was higher than those reported by Renken and Warthesen (1993) and Saffert et al. (2008  74  VD content for outside bottles (% Control) Vitamin D retention  100% 90% 80%  70% 60% 50% 40% 30% 20% Day 1  Day 8  Day 15  Day 22  Storage time Crystalline D3  SDS D3  CWS D3  AD premix  VD content for middle bottles (% Control)  Vitamin D retention  100% 90% 80% 70% 60%  50% 40% 30% 20% Day 1  Day 8  Day 15  Day 22  Storage time Crystalline D3 Figure 4.6  SDS D3  CWS D3  AD premix  Vitamin D retention as a function of the interaction of formulation and  exposure time at different positions.  75  Table 4.6  Vitamin A content (IU/100 mL) in skimmed milk fortified with vitamin A  and D exposed to light of 2000 lux or stored in the dark (n=2). Vitamin D formulation  Day 8  Day 22  Light exposed  In the dark  Light exposed  In the dark  Crystalline D3  0.0  9.8  0.0  11.4  SDS D3  0.0  16.8  0.0  9.9  CWS D3  0.0  20.3  0.0  7.3  12.0  206.3  0.0  185.2  AD premix  76  Table 4.7  Vitamin A content (IU/100 mL) in skimmed milk fortified with vitamin A or  vitamin A and D, and stored in the dark (n=2). Milk fortification  Initial concentration After 1 week  After 3 weeks  Vitamin A only  220.6  49.3  6.5  CWS D3 and Vitamin A  206.3  44.3  7.3  Crystalline D3 and vitamin A  217.7  54.0  9.3  77  and 2009). Whether the rapid degradation of vitamin A accelerated the degradation of vitamin D remains to be confirmed. Bartholomew and Ogden (1990) suggested that added vitamin A might be more sensitive to light than the indigenous vitamin A but commercial emulsifiers might improve the stability of fortified vitamin A since it could provide as good protection as the milk fat. In the present research, the AD premix was an emulsified form containing polysorbate 80 and polyglycerol monooleate as emulsifiers. This may enhance the stability of vitamin A in AD premix compared to the water dispersible form when being stored in the dark. However, both vitamin A forms were not stable upon light exposure. 4.7  Conclusion Commercial vitamin D3 formulations did show different stability to the light  exposure in skimmed milk. Significant vitamin D losses were observed for all milk samples being exposed to light. Vitamin D retention was not associated with the position of the sample container although the center container had less area being exposed to light than outside container. The added water dispersible vitamin A and the vitamin A in AD premix degraded completely on light exposure after 22 days storage. However, the water dispersible vitamin A degraded significantly even when being stored in the dark, while the vitamin A content in AD premix was retained at more than 90% of the original concentration in the dark. More research needs to be done to determine whether the degradation of vitamin A may have an impact on vitamin D stability.  78  Chapter 5: Thermal stability of vitamin D2 and vitamin D3 solutions at pH 3.5 and 6.6 Both vitamin D2 and D3 can be used for food fortification despite the debate on potency of these two forms. The Food and Drug Regulations (2012) does not specify which form of vitamin D should be used for food fortification. Although usually vitamin D3 is often added to milk products, milk fortified with vitamin D2 has also been reported. Vitamin D2 might also be used for fortification for some milk substitutes or other beverages, such as soy milk, almond milk, and orange juice. Studies in the pharmaceutical field indicated that in solid form, vitamin D2 and D3 demonstrated different stability under various conditions (Byrn, 1976; Grady & Thakker, 1980). However there are few studies published for the stability comparison of the two vitamin D forms in liquid solution or foods. In this research, a model system at typical pH of orange juice and milk was set up to compare the thermal stability of vitamin D2 and D3. Crystalline vitamin D2 and D3 content in buffer of pH 3.5 and 6.6 were analyzed before and after thermal treatment of 72°C for 15 seconds since this condition is typical for milk pasteurization (CFIA, 2010). The experiment design is shown in Figure 5.1. 5.1  Materials Buffer solutions with pH 3.5 and pH 6.6 were used to test the thermal stability of  vitamin D2 and D3. Sodium phosphate dibasic (0.2 M) and citric acid (0.1 M) were used to prepare the buffer. The buffer pH was adjusted to the specific level using a Corning Pinnacle M530 pH meter (Gold River, CA). Crystalline vitamin D2 (E5750) and D3 (C9756) from Sigma-Aldrich (Oakville, ON) were used to make methanol stock solution  79  Pre-heated buffer, 72 °C  Ambient buffer  Vitamin D addition  Vitamin D and internal standard addition  Hold for 15 seconds  Ice bath, internal standard addition  Vitamin D measurement  Vitamin D measurement  Vitamin D retention calculation  Figure 5.1 Overview of study design for stability of vitamin D2 and D3 to thermal treatment in a model system.  80  of 200 IU/mL and 160 IU/mL respectively. For the purpose of quantification, vitamin D2 was used as internal standard for vitamin D3 and vitamin D3 was internal standard for vitamin D2 determination. 5.2  Thermal processing Three glass tubes each containing 4 mL of pH 3.5 or pH 6.6 buffer were heated up  to 72 °C using water bath (Blue M, Blue Island, IL). A thermometer was placed in a fourth tube to monitor the temperature of the buffer. An 80 µl aliquot of vitamin D2 or 100 µl of vitamin D3 (ca. 16 IU) was added into the pre-heated buffer solution without taking the tubes from the water bath. This amount of vitamin D gives a similar final concentration as vitamin levels in fortified milk. After holding the temperature for 15 seconds, the tube was removed and placed into an ice bath immediately. Then 80 µl of vitamin D2 or 100 µl of vitamin D3 (ca. 16 IU) was added as an internal standard into the tube in ice bath. The thermal treatment was conducted in three replicates. Another set was held at ambient temperature as controls. 5.3  Vitamin D analysis The vitamin D was extracted and analyzed on HPLC using a similar protocol  described in section 3.2 except that saponification and SPE purification steps were not necessary. The vitamin D retention was calculated by comparing the vitamin D content in the thermal treated milk with that in the unheated milk. The retentions were processed using two-way ANOVA. 5.4  Statistical analysis  Two-way ANOVA was conducted using Minitab 16 (State College, PA) to investigate  81  the difference in the vitamin D retention between vitamin D2 and D3 in pH 3.5 and 6.6 buffers. 5.5  Results and discussion Over 90% retention of vitamin D was observed after thermal treatment for both  forms of vitamin D (Table 5.1). Only the treatment at pH 6.6 of vitamin D3 showed a significant difference (P < 0.05) when comparing with the corresponding unheated sample using Tukey's test. However, the difference was less than 8%. The results of ANOVA are shown in Table 5.2. Two way ANOVA indicated that there was no significant difference between vitamin D2 and D3 samples in vitamin D retention after thermal treatment. The thermal stability of vitamin D in pH 3.5 versus pH 6.6 buffer was also not significantly different . These results are similar to what Mattila et al. (1999) found in house cooking process of egg, fish and mushroom. 5.6  Conclusion Vitamin D2 and D3 showed a similar thermal stability at pH 3.5 and 6.6 to High  Temperature Short Time (HTST) treatment of 72 °C for 15 seconds. The total vitamin D loss was less than 8%. For the purpose of fortification, both vitamin D2 and D3 can be added to liquid food at pH range of 3.5 to 6.6 since the two forms of vitamin D showed similar stability. Both forms can be added before the HTST pasteurization since this process showed little impact on vitamin D content.  82  Table 5.1  The impact of thermal treatment (72 °C for 15 seconds) on vitamin D content  in pH 3.5 and pH 6.6 buffers. Values are mean values ± standard deviation (SD, n=3) or range (n=2). Vitamin D content (IU) Vitamin D form  Unheated (n=2)  72°C 15"(n=3)  Vitamin D  and pH of buffer  Mean  ±  Range  Mean  ±  SD  retention %  Vitamin D3, pH 3.5  15.39  ±  0.19  15.01  ±  0.46  97.54%  Vitamin D2, pH 3.5  16.70  ±  0.62  15.74  ±  0.46  94.23%  Vitamin D3, pH 6.6  16.79  ±  0.38  15.51  ±  0.20  92.32%  Vitamin D2, pH 6.6  16.05  ±  0.52  15.28  ±  0.72  95.15%  83  Table 5.2 The result of two-way ANOVA for vitamin D2 and D3 retention after thermal treatment (72 °C for 15 seconds) at pH 3.5 or pH 6.6. Factors  DF  Seq SS  Adj SS  Adj MS  F value  Probability  pH  1  0.001  0.001  0.001  1.46  0.262  Vitamin D form  1  0.000  0.000  0.000  0.02  0.896  pH * vitamin D form  1  0.003  0.003  0.003  2.96  0.123  Error  8  0.008  0.008  0.001  Total  11  0.012  84  Chapter 6: General discussion and conclusions The present research investigated the vitamin D content in 104 cartons of fluid milk samples sold in Vancouver between November 2011 and September 2012. Fortification non-compliance was identified in 58% of the sampled milk using 35.2 to 46.9 IU/100 mL as the criteria for compliance. The fortification compliance of vitamin D in fluid milk in the present research was compared with the investigation on BC milk reported in 1993 by Chen et al. For the purpose of comparison, 20% margin of tolerance relative to the claimed fortification level was applied to evaluate the compliance (Figure 6.1). After 19 years, there was an obvious improvement in vitamin D fortification of milk. The percentage of samples containing 80-120% of the claimed vitamin D amount increased from 27% to 65%. However, 27% of the sampled milks sampled in 2012 are under-fortified, which is the same percentage of under-fortification reported for the 15 milks sampled from British Columbia as reported by Chen et al. in 1993. Wide variation was found among different brands and between skim versus 2% or 3.25% milk. Vitamin D content was also found to fluctuate between milk sampled at different times. All three factors (brand, fat content and sampling set) were significant contributors to the vitamin D variation. Some possible reasons are summarized in Table 6.1. More research needs to be done to understand the causes of the recurring underfortification. Of seven brands sampled in the present research, vitamin D in skim milk from brand 5 and 7 were consistently low. These two brands were produced by the same dairy processor. Consistently low vitamin D content may indicate a systematic  85  1993 n=15, BC  2012 n=52, Vancouver  10% 27%  <80% of label value 80-120% of label value  63%  Figure 6.1  27% 47%  >120% of label value  27%  The percentages of milk samples categorized as under-fortified, over-  fortified or satisfying the fortification target for vitamin D (based on a 20% margin of tolerance) in the present study (2012) compared to the study by Chen et al. (1993).  86  Table 6.1  Possible reasons contributing to the variation in vitamin D content in  sampled milk. Significant Factor  Possible reasons for the variation in vitamin D content  Brand  Different vitamin formulation Different vitamin addition method (injection or batch addition) Different processing procedure for vitamin addition Different storage condition for both dry vitamin and working solution Different packaging  Milk fat content  Possible protection provided by milk fat Different vitamin formulation used for milk with different fat content considering the solubility of vitamin D  Sampling set  Poor quality management Degradation of vitamin working solution during storage Any changes in the production (formulation or fortification procedure) may change the final concentration of vitamin D  87  mistake in the vitamin D addition protocol from this processor. For example, the calculation of vitamin D addition might be incorrect because of inaccurate concentration information from vitamin supplier or simply using wrong calculation. Since vitamin A needs to be added into skim milk as well, the vitamin D formulation used for skim milk is usually different from that used for the 3.25% milk. Low vitamin D content might also be caused by the low solubility of the fat soluble vitamin formulations in skim milk. The investigation in North Carolina (Hicks et al., 1996) showed vitamin D was added at different processing steps in different plants and different storage conditions were applied on vitamins. The impact of milk processing on vitamin D is not well studied. However, Bartholomew and Ogden (1990) suggested that the homogenization step improved the blending of fat soluble vitamin with milk solid and milk fat, which acted as natural emulsifiers and provided protection. Bartholomew and Ogden (1990) also reported that blending vitamin A into milk fat after separation was more effective than adding vitamin A as a highly concentrated solution. Since vitamin D is also a fat soluble vitamin, different addition points and solution preparation might introduce variation into the final concentration in milk. Besides the processing procedure, vitamin D formulations also have different stability, as indicated in Chapter 4. If different vitamin formulations are applied in milk fortification by milk processors, the vitamin D concentration of final products may be variable as well. The storage conditions also affect the stability of both dry and emulsified vitamin D. If processing and formulation information could be collected from processors, it would be helpful to understand why wide variation of vitamin D content existed in milk products. 88  In the present research, only fluid milk of Vancouver was included. To understand the national trend, milk with different fat content of different areas should be analyzed. The stability to light exposure of four commercial vitamin D formulations was found to be different. A well encapsulated formulation may reduce the vitamin D loss in milk during storage. The impact of light exposure on vitamin D in fluid milk has been reported previously (Renken & Warthesen, 1993; Saffert et al., 2008 and 2009). Although different milk samples and storage conditions were applied in these studies, significant vitamin D losses were observed in milk when being exposed to light. The results of previous studies and the present study are summarized in Table 6.2. In the study of Saffert et al. (2008), unfortified milk samples were studied and thus only indigenous vitamin D was present. In the other three studies, milk samples were fortified with vitamin D at concentration ranging from 40 to 48 IU/100 mL. The vitamin D loss in the study reported by Saffert et al. (2009) at the end of week 4 was much lower than that at the end of week 3 in the present research, despite higher storage temperature. By the end of week 12, the total exposure reached 1092 hours, much longer than 504 hours in the present research. However the vitamin D loss is still lower. The lower light intensity of 700 lux might be the reason. In the study of Saffert et al. (2008), after 1092 hours exposure at 3000 lux, the vitamin D loss also remained lower than the present research. For both studies of Saffert et al. (2008 & 2009), the details of vitamin D formulation and fortification protocol were not given. A different vitamin D formulation might have been used and affected the vitamin D loss.  89  Table 6.2  Comparison of vitamin D loss in milk exposed to light during storage.  Initial content (IU/100 mL)  Vitamin D loss  Reference  Sample and package  Vitamin D  Vitamin A  Storage conditions  Week 2  Week 3  Week 4  Week 12  The present  Pasteurized skimmed milk  40  200  2000 lux, 24  25-60%  37-71%  NA *  NA  10%  NA  33%  65%  NA  NA  a - 14%  a - 39%  study  hrs/day, 4°C  500 ml in glass bottle  Saffert et  UHT 1.5% fat milk  46 - 48  675-739  700 lux, 13 hrs/day,  al.,. 2009  1 litre in clear plastic jug  Saffert et al.,  Unfortified UHT whole  2008  milk  b - 1700 lux  b - 12%  b - 49%  1 litre in clear plastic jug  c - 3000 lux  c - 24%  c - 57%  NA  NA  23°C  0.49  110  a - 700 lux  13 hrs/day, 23°C Renken &  Pasteurized skim milk  Warthesen,  a - 1.8 litre in plastic jug  1993  b - 30 ml in 20×150 mm  42  Unknown  3229 lux, 24  a - 25% NA  hrs/day, 4°C  b - 50%  glass tube  *NA = not applicable  90  The storage condition of the present study is close to that of Renken and Warthesen's research. Light exposure was applied in a 24 hours/day manner and skim milk was fortified at a similar concentration. Plastic containers and glass tubes were used in the research of Renken and Warthesen. The plastic container might provide better protection than the clear glass containers used in the present research. The vitamin D loss in glass tubes was expected to be higher than that in the 500 mL glass bottle of the present research since higher light intensity was applied. However, the vitamin D loss was slightly lower than that of the present research at the end of week 2 although higher light intensity was applied. Again, different vitamin D formulation might have been used. In the present research, the vitamin D stability was not different between the center bottle and outside bottle despite more surface area being exposed to light in the outside bottles. Saffert et al. (2008) found that the vitamin D loss in unfortified milk held in clear packages was independent of the light intensity. However, the initial vitamin D content was so low in unfortified milk, the conclusion of Saffert et al. (2008) may not be applicable to the degradation of vitamin D in fortified milk. The instability of vitamin D to light is caused by the singlet oxygen produced by a photo-sensitizer (Renken and Warthesen, 1993; King & Min, 1998 & 2002). As a photosensitizer, riboflavin can produce singlet oxygen on the exposure of light (King & Min, 1998 & 2002). According to U.S. Department of Agriculture, Agricultural Research Service (2012), the riboflavin content in 100 g skim milk is about 0.182 mg. A clear package, either plastic or glass, with high transmittance of light, may provide enough energy for riboflavin to produce adequate singlet oxygen and start the oxidation of  91  vitamin D, and thus no significant difference of vitamin D loss was found between bottles despite the different light intensity being applied and different exposure area. As mentioned in section 4.6, vitamin A in fortified skimmed milk was completely degraded after storage with light exposure. Crank and Pardijanto (1995) suggested that the photosensitized oxidation of vitamin A is initiated by singlet oxygen. Since riboflavin produces singlet oxygen on light exposure, its presence in the milk may accelerate the degradation of vitamin A. However, whether the degradation of vitamin A affects the stability of vitamin D or not still remains unknown. If the degradation of vitamin A also has an impact on vitamin D stability, then the different amount of vitamin A in the four studies of Table 6.2 may also have caused the different vitamin D loss. To confirm this, more research needs to be done. As a conclusion, non-compliance, especially under-fortification, is still a problem of vitamin D fortification of milk in Vancouver. Both vitamin D2 and D3 are stable during HTST treatment in liquid system at pH 3.5 or 6.6. Different vitamin D formulations differ in their stability to light. Encapsulated vitamin D with good protection may reduce the vitamin D loss on light exposure. The present research only analyzed skim and 2% or 3.25% milk. To better understand the vitamin D variation in commercial milk, analysis of vitamin D needs to be conducted on milk products with different fat content from different brands across the country. Considering the fluctuating vitamin D content between different lots, information about the fortification protocols provided by milk processors would be helpful to screen the possible sources of lot-to-lot variation. However, such information  92  might be very difficult to obtain from the industry since many processors consider processing information confidential. The light stability of different vitamin D formulations were only tested in skimmed milk. Whether the stability might be affected by fat content is still unclear despite higher vitamin D content was found in 3.25% commercial milk than in skim milk. The potential protection provided by milk fat still needs to be confirmed by further research. The possible impact of vitamin A degradation on vitamin D also needs to be investigated. Based on the results obtained in the present research, using formulation with higher stability or avoiding light exposure during storage would improve the retention of vitamin D content in milk product and thus may help to improve the compliance of fortification. Vitamin D2 showed similar thermal stability as vitamin D3 at pH 3.5 and 6.6. Both vitamin D forms are suitable for the fortification of food system at these pH values from the point of stability under pasteurization.  93  References Amberg-Schwab, S., Weber, U., Burger, A., Nique, S., & Xalter, R. (2006). Development of passive and active barrier coatings on the basis of inorganic–organic polymers. Monatshefte für Chemie - Chemical Monthly, 137, 657-666. Armas, L. A. G., Hollis, B. W., & Heaney, R. P. (2004). Vitamin D2 is much less effective than vitamin D3 in humans. Journal of Clinical Endocrinology & Metabolism, 89, 5387-5391. Ball, G. F. M. (2006). Vitamins in foods: Analysis, bioavailability, and stability. 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Journal of Steroid Biochemistry and Molecular Biology, 103, 626-630.  101  Appendices Appendix A Time and places for commercial milk sampling and analysis  Brand & Type  Set #  Date of purchase  Brand 1 skim (Processor A)  Set 1 Set 2 Set 3 Set 4 Set 1 Set 2 Set 3 Set 4  23-Nov-11 10-Jan-12 28-Feb-12 9-Aug-12 28-Dec-11 23-Jan-12 29-Feb-12 28-Aug-12  28-Nov-11 17-Jan-12 7-Mar-12 22-Aug-12 13-Jan-12 8-Feb-12 7-Mar-12 12-Sep-12  5 7 8 13 16 16 7 15  Safeway at Sasamat St & 10th Ave Safeway at Sasamat St & 10th Ave Safeway at Mcdonald St & Broadway Safeway at Sasamat St & 10th Ave T&T at China Town Donald's Market at East Hasting Save on Food at UBC Save on Food at UBC  Brand 2 skim Set 1 (Processor unknown) Set 2 Set 3 Set 4 Brand 2 3.25% Set 1 (Processor unknown) Set 2 Set 3 Set 4  25-Nov-11 10-Jan-12 N/A N/A 25-Nov-11 24-Jan-12 N/A N/A  1-Dec-11 19-Jan-12 N/A N/A 8-Dec-11 2-Feb-12 N/A N/A  6 9 N/A N/A 13 9 N/A N/A  Safeway at Commercial Drive & Broadway Safeway at Commercial Drive & Broadway N/A N/A Safeway at Mcdonald St & Broadway Safeway at Mcdonald St & Broadway N/A N/A  Brand 1 3.25% (Processor A)  Date of analysis  Period of storage (day)  Place of procurement  102  Appendix A Time and places for commercial milk sampling and analysis (continued)  Brand & Type  Set #  Date of purchase  Brand 3 skim (Processor B)  Set 1 Set 2 Set 3 Set 4 Set 1  24-Nov-11 18-Jan-12 14-Mar-12 16-Aug-12 25-Nov-11  5-Dec-11 30-Jan-12 26-Mar-12 27-Aug-12 9-Dec-11  11 12 12 11 14  (Processor B)  Set 2 Set 3 Set 4  11-Jan-12 21-Feb-12 16-Aug-12  23-Jan-12 27-Feb-12 30-Aug-12  12 6 14  Shoppers Drug Mart at UBC Shoppers Drug Mart at UBC Shoppers Drug Mart at UBC Shoppers Drug Mart at UBC Shoppers Drug Mart at UBC Shoppers Drug Mart at Broadway & Commercial Drive Shoppers Drug Mart at UBC Shoppers Drug Mart at UBC  Brand 4 skim (Processor C)  Set 1 Set 2 Set 3 Set 4 Set 1 Set 2 Set 3 Set 4  5-Dec-11 23-Jan-12 24-Feb-12 9-Aug-12 30-Nov-11 27-Jan-12 24-Feb-12 9-Aug-12  14-Dec-11 1-Feb-12 8-Mar-12 23-Aug-12 13-Dec-11 8-Feb-12 8-Mar-12 23-Aug-12  9 9 13 14 13 12 13 14  Donald's Market at East Hastings Donald's Market at East Hastings Donald's Market at East Hastings Donald’s Market at East Hastings Donald's Market at East Hastings Donald's Market at East Hastings Donald's Market at East Hastings Donald’s Market at East Hastings  Brand 3 3.25%  Brand 4 3.25% (Processor C)  Date of analysis  Period of storage (day)  Place of procurement  103  Appendix A Time and places for commercial milk sampling and analysis (continued)  Brand & Type  Set #  Date of purchase  Brand 5 skim (Processor D)  Set 1 Set 2 Set 3 Set 4 Set 1 Set 2 Set 3 Set 4  29-Nov-11 7-Feb-12 21-Feb-12 9-Aug-12 30-Nov-11 21-Jan-12 7-Mar-12 31-Aug-12  12-Dec-11 20-Feb-12 28-Feb-12 20-Aug-12 13-Dec-11 3-Feb-12 20-Mar-12 13-Sep-12  13 13 7 11 13 13 13 13  Safeway at Sasamat St & 10th Ave Safeway at Mcdonald St & Broadway Safeway at Commercial Drive & Broadway Safeway at Sasamat St & 10th Ave Safeway at Sasamat St & 10th Ave Safeway at Commercial Drive & Broadway Safeway at Commercial Drive & Broadway Safeway at Commercial Drive & Broadway  Set 1 * Set 2 Set 3 Set 4 Set 1 Set 2 Set 3 Set 4  15-Dec-11 30-Jan-12 25-Feb-12 16-Aug-12 21-Dec-11 5-Feb-12 2-Mar-12 16-Aug-12  22-Dec-11 10-Feb-12 9-Mar-12 7-Sep-12 16-Jan-12 27-Feb-12 19-Mar-12 10-Sep-12  7 11 13 22 26 22 17 25  Safeway at Commercial Drive & Broadway Safeway at Mcdonald St & Broadway Safeway at Commercial Drive & Broadway Safeway at Commercial Drive & Broadway Safeway at 4th Ave near Kitsilano Superstore at Grandview Highway Safeway at Commercial Drive & Broadway Safeway at Commercial Drive & Broadway  Brand 5 3.25% (Processor D)  Brand 6 skim (Processor E)  Brand 6 2% (Processor E)  Date of analysis  Period of storage (day)  Place of procurement  104  Appendix A Time and places for commercial milk sampling and analysis (continued)  Brand & Type Brand 7 skim (Processor D)  Set #  Date of purchase  Set 1 29-Jan-12 Set 2 3-Feb-12 Set 3 17-Mar-12 Set 4 16-Aug-12 Brand 7 3.25% Set 1 29-Jan-12 (Processor D) Set 2 3-Feb-12 Set 3 24-Feb-12 Set 4 24-Aug-12 * 2-L package was sampled instead of 1 L.  Date of analysis  Period of storage (day)  Place of procurement  7-Feb-12 14-Feb-12 27-Mar-12 27-Aug-12 10-Feb-12 14-Feb-12 9-Mar-12 4-Sep-12  9 11 10 11 12 11 14 11  Superstore at Grandview Highway No Frill at Broadway Superstore at Grandview Highway Superstore at Grandview Highway Superstore at Grandview Highway No Frill at Broadway Superstore at Grandview Highway Superstore at Grandview Highway  105  Appendix B Vitamin D content of commercial fortified fluid milks and t-test for two containers of same product in each set. Set 1 Carton A  B  Rep.  Set 2  Set 3  Set 4  Set 1  Brand 1 - Skim 31.98  36.93  1.37  35.16  28.16  29.48  26.44  26.75  2  30.98  37.12  1.48  35.77  30.29  29.39  25.98  27.04  3  31.32  36.76  1  34.61  35.56  1.18  34.72  31.24  28.98  25.98  26.46  2  31.66  35.14  1.20  34.51  30.49  29.63  24.77  26.90  3  32.63  37.62  NS*  NS  NS  NS  28.58  28.04 NS  NS  NS  Brand 2 - 3.25%  1  22.00  31.27  39.20  36.13  2  22.51  31.78  40.75  35.42  3  23.46  31.22  38.27  1  23.67  31.13  38.20  35.02  2  23.70  30.14  40.37  36.95  3  22.66  30.57  39.73  NS  NS  NS  T-test  Brand 3 - skim A  B  T-test  Set 4  Brand 1 - 3.25%  Brand 2 - skim  B  Set 3  1  T-test  A  Set 2  NS Brand 3 - 3.25%  1  45.34  34.54  36.28  31.76  42.15  42.14  29.30  31.64  2  46.53  33.91  38.01  31.72  43.24  43.81  28.71  30.19  3  45.86  42.97  43.74  1  45.04  32.12  36.59  30.10  43.08  42.58  24.96  28.94  2  44.84  32.52  36.45  31.42  44.83  41.69  26.93  30.36  3  45.74  42.84  43.32  NS  NS  NS  NS  NS  NS  NS  NS  106  Appendix B Vitamin D content of commercial fortified fluid milks and t-test for two containers of same product in each set (continued). Set 1  Set 2  Set 3  Set 4  Set 1  Brand 4 - skim A  B  Set 4  Brand 4 - 3.25%  34.91  32.68  40.41  44.70  39.55  42.81  35.82  57.77  2  33.99  31.25  41.60  43.37  41.31  41.40  36.33  57.18  3  34.64  1  34.73  31.08  40.65  45.41  39.98  41.77  36.75  57.21  2  34.20  29.92  40.59  44.57  41.90  41.14  33.12  56.85  3  34.51  NS  NS  NS  NS  41.36  41.54 NS  NS  NS  NS  Brand 5 - skim  B  Set 3  1  T-test  A  Set 2  Brand 5 - 3.25%  1  15.80  13.81  9.59  14.70  30.49  46.38  36.04  41.08  2  15.84  14.93  9.57  11.24  32.88  46.94  35.44  40.90  3  15.46  1  14.00  13.98  10.13  14.32  32.08  47.59  38.89  41.73  2  14.90  13.80  10.35  14.45  32.63  48.34  36.15  41.36  3  14.58  p=0.05  NS  NS  NS  T-test  32.25  33.14 NS  NS  NS  NS  Carton Rep. A  B  T-test  Brand 6 - skim  Brand 6 - 2%  1  32.06  38.86  33.17  38.02  38.70  34.44  35.63  41.79  2  34.01  38.30  34.16  40.32  41.82  33.97  35.83  39.16  3  32.27  1  31.67  38.08  34.75  39.23  37.94  33.00  35.64  40.26  2  32.24  39.89  34.99  39.52  38.24  34.18  35.37  40.71  3  34.34  NS  NS  NS  NS  38.11  37.11 NS  NS  NS  NS  107  Appendix B Vitamin D content of commercial fortified fluid milks and t-test for two containers of same product in each set (continued). Set 1  Set 2  Set 3  Set 4  Set 1  Brand 7 - skim A  B  T-test  Set 2  Set 3  Set 4  Brand 7 - 3.25%  1  11.96  11.83  8.27  14.15  44.29  14.49  36.12  39.22  2  13.01  11.90  8.60  14.18  45.57  12.73  36.14  37.09  1  11.30  11.25  8.92  14.56  44.61  14.54  34.88  38.26  2  11.86  11.89  8.34  14.45  46.08  13.20  33.83  37.53  NS  NS  NS  NS  NS  NS  NS  NS  108  Appendix C Vitamin formulations used in light exposure study.  109  Appendix C Vitamin formulations used in light exposure study (continued).  110  Appendix C Vitamin formulations used in light exposure study (continued).  111  Appendix C Vitamin formulations used in light exposure study (continued).  112  Appendix C Vitamin formulations used in light exposure study (continued).  113  Appendix C Vitamin formulations used in light exposure study (continued).  114  Appendix C Vitamin formulations used in light exposure study (continued).  115  Appendix C Vitamin formulations used in light exposure study (continued).  116  Appendix C Vitamin formulations used in light exposure study (continued).  117  

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