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Comparison of Consumer Acceptance, Physico-chemical Properties, and Bioactive Delivery of Blueberry Extract and Whole Blueberry Powder Confections

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in

the Graduate School of The Ohio State University

Meredith Ruth Myers

Graduate Program in Food Science and Technology

The Ohio State University

2018

Thesis Committee

Yael Vodovotz Ph.D., Advisor

Christopher Simons, Ph.D.

Copyrighted by

Meredith Ruth Myers

2018

Abstract

Advances in chemotherapy have greatly increased the rate of survival among many of the most common cancers, but these improvements in cancer treatment have also caused new side effects in patients that greatly affect their daily lives. One of the most common of these secondary side effects is chemotherapy-induced cognitive impairment

(CICI) also known colloquially in the medical community as “chemo brain”. CICI affects up to 78% of cancer patients who have received chemotherapy, and there are no current treatments available for the associated cognitive side effects which include memory loss, depression, and general feelings of mental “fogginess”. The consumption of diets rich in fruits and vegetables have been implicated with multiple health benefits, with the consumption of anthocyanin rich foods, such as blueberries, showing particular promise in the area of improvement of cognitive health. The overall objective of this work was to develop and fully characterize two stable delivery vehicles for blueberry bioactives with regards to consumer acceptance, physico-chemical properties, and bioactive delivery in order to use in future clinical studies looking at CICI. A secondary objective of this work was to directly examine how the inclusion of an anthocyanin-rich blueberry extract

(BBEC) vs. a lyophilized whole fruit blueberry powder (BBPC) would affect consumer acceptance, physico-chemical properties, and bioactive delivery of an equivalent

ii functional food matrix, which would have useful applications in a broader sense of functional food product development.

In this work two functional gelatin confections were successfully developed and were found to be promising candidates for use in future CICI clinical trials because of their stability over one month of refrigerated storage. It was found that the main differences between including a whole blueberry powder vs. an anthocyanin-rich blueberry extract within an equivalent gelatin confection matrix were bioactive retention, gel strength, texture, and freezable water content, but with no significant differences seen in their consumer acceptability. BBPC was found to retain a significantly higher number of anthocyanins post- confection processing, produce a harder gel micro structure, require a one-week texture stabilization period, as well as contain a higher %FW when compared to the BBEC formula.

BBPC and BBEC were compared in a dietary intervention where healthy men and women were fed a single equivalent acute dose and their 24-hour urine collected and analyzed. Results showed that all participants complied with their 7-day low berry washout diets, and that BBEC and BBPC were able to deliver approximately 0.05% of the anthocyanins they contained post processing and pre-ingestion. Importantly, it was also observed that there was not a significant difference in the anthocyanin delivery between BBEC and BBPC when controlling for inter individual metabolic heterogeneity.

There were also no significant differences seen in product acceptability between the

BBEC and BBPC when tested in the clinical rather than the consumer setting.

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These studies show the importance of considering bioactive source when developing functional food products for use in nutritional clinical studies. Furthermore, these studies demonstrate the potential to successfully develop two functional confections for the delivery of blueberry anthocyanins for future use in CICI and cognitive health clinical studies. Larger clinical studies in a CICI cohort is necessary to test effectiveness of blueberry anthocyanins to mitigate cognitive symptoms associated with the disorder.

Dedication

This work is dedicated to my mother and father, for always cultivating my curiosity and

love of learning.

Acknowledgments

I would like to start by thanking my advisor, Dr. Yael Vodovotz for all of the guidance she has provided me throughout my time at The Ohio State University in the

Department of Food Science and Technology. From exposing me to research early on as an honors undergraduate student, to taking me on as a graduate student in her laboratory for my Master’s degree, I am forever thankful for all of the patience, encouragement, and laughs you have provided over these past six years. I can only hope that my future bosses will be as supportive of me as you have been. I also would like to thank my Masters

Committee member, Dr. Christopher Simons, for being a wonderful professor and teaching some of my favorite classes during my time as a Masters student, as well as being fantastic career role model, I hope our professional paths will continue to cross in the future.

Many thanks also go out to four special past Vodovotz lab students/ postdocs, Dr.

John Frelka, Dr. Sravanti Paluri, Tim Vazquez, and Dr. Jennifer Ahn-Jarvis, all of whom made me the researcher I am today, as well as immensely helped with the blueberry confection shelf-study. Whether it was training me on our lab’s instruments, answering questions about statistics and data analysis, or helping me edit multiple presentations and posters, you all taught me the art of weaving a great scientific narrative that is both compelling and technically sound.

Thank you to Dr. Beth Grainger, Dr. Steven Clinton, all of the nurses at the

Clinical Research Center, and my clinical study participants for helping me with my IRB and giving me a crash course in clinical trial design and execution. It was definitely a unique opportunity for a food scientist, and I will carry with me the skills and appreciation for those that work in clinical settings that this project has taught me forever.

Thank you to all of the Vodovotz lab members, undergraduate researchers, visiting scholars, and Metro students who were integral in the execution of my research projects, and have helped me develop my own management skills. I would also like to thank all of my friends in office 240 and throughout Parker Food Science, who with our many lunches and games of GeoGuessr made these past two years of work a lot more fun. Lastly, thank you to two my best friends, Katie Williamson and Mackenzie Hannum, my twin sister Grace, and the rest of my family, I can honestly say I could not have completed this graduate degree without all of your encouragement.

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Vita

May 2012…………………………………….Ursuline Academy, Cincinnati, OH

May 2016……………………………………. B.S. Food Science and Technology, The

Ohio State University, Columbus. OH

August 2016 to present……………………….Graduate Research Associate, Department

of Food Science and Technology, The

Ohio State University, Columbus, OH

Fields of Study

Major Field: Food Science and Technology

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Table of Contents

Abstract ...... ii Dedication ...... v Acknowledgments...... vi Vita ...... viii List of Tables ...... xii List of Figures ...... xiii List of Equations ...... xv Chapter 1. Introduction ...... 1 1.1 Statement of Problem ...... 1 1.2 Hypotheses ...... 2 Aim 1: ...... 3 Aim 2: ...... 3 Chapter 2. Literature review ...... 5 2.1 Blueberries ...... 5 2.2 Commercial production and consumption of blueberries ...... 6 2.2.1 U.S. Blueberry production ...... 6 2.2.2 U.S. Blueberry consumption ...... 6 2.3 Blueberry components and stability...... 7 2.3.1 Major blueberry components ...... 7 2.3.2 Anthocyanin profile of blueberries ...... 8 2.3.3 Stability of Anthocyanins to processing and storage ...... 11 2.4 Absorption, metabolism and bioavailability of anthocyanins...... 13 2.4.1 Absorption of anthocyanins in humans...... 13 2.4.2 Metabolism of anthocyanins in humans ...... 15 2.4.3 Pharmacokinetics of anthocyanins in humans ...... 16 2.5 Anthocyanins and health ...... 17

2.6 Anthocyanins and neuroprotection ...... 19 2.7 Blueberries and cognitive dysfunction...... 22 2.8 Chemotherapy-induced cognitive dysfunction (CICI)...... 24 2.9 Dietary interventions for cognitive improvement ...... 26 2.10 Background of food gels ...... 27 2.11 Confectionary gels ...... 28 2.11.1 Role of water in confectionary gels ...... 29 2.12 Gelatin ...... 29 2.12.1 Gelatin composition ...... 30 2.12.2 Gelatin gelation ...... 31 Chapter 3. Comparison of consumer acceptance and physico-chemical properties of blueberry extract and whole blueberry powder confections designed for a future clinical trial ...... 35 3.1 Introduction ...... 35 3.2 Materials and Methods ...... 38 3.2.1 Blueberry confection preparation ...... 38 3.2.2 Thermogravimetric Analysis (TGA)...... 40 3.2.4 Texture Profile Analysis ...... 42 3.2.5 Rheological Analysis ...... 42 3.2.6 Bioactive Identification and retention ...... 43 3.2.7 Sensory analysis of blueberry confections ...... 46 3.2.8 Statistical analysis ...... 47 3.3 Results and Discussion ...... 48 3.3.1 Anthocyanin identification, quantification, and retention after confection manufacturing and 4-week storage ...... 48 3.3.2 Differences in water-binding of blueberry extract and lyophilized blueberry powder confections ...... 53 3.3.3 Microstructural and macroscopic texture differences observed in blueberry extract and powder confections over one month storage ...... 56 3.3.4 Sensory analysis of blueberry confections ...... 62 3.4 Conclusion ...... 65 Chapter 4. Comparison of anthocyanin delivery after consumption of blueberry extract and whole blueberry powder confections in healthy men and women ...... 68 4.1 Introduction ...... 68 x

4.2 Materials and Methods ...... 70 4.2.1 Blueberry confection preparation ...... 70 4.2.2 Anthocyanin delivery clinical study ...... 72 4.2.3 Sensory analysis ...... 74 4.2.4 Dietary control and analysis...... 75 4.2.5 Anthocyanin urinary analysis ...... 76 4.2.6 Statistical Methods ...... 77 4.3 Results and discussion ...... 77 4.3.1 Participants ...... 77 4.3.2 Sensory analysis of blueberry confections ...... 79 4.3.3 Anthocyanin levels in urine ...... 83 4.4 Conclusion ...... 86 Chapter 5. Conclusions and future work ...... 87 Bibliography ...... 90 Appendix A: Blueberry gummy clinical documents ...... 103 A1. Study informed consent form ...... 103 A2. Blueberry gummy clinical informational flyer ...... 110 A3. Blueberry gummy clinical scripts ...... 111 A4. Blueberry Gummy Participant Health History Form ...... 113 A5. Berry- restricted diet ...... 117 A6. Three Day Dietary Record ...... 118 A7. Blueberry gummy clinical sensory questionnaire ...... 120 A8. Blueberry gummy 24- hour urine instructions ...... 127 A9. Nutrient consumption breakdown of clinical participants ...... 128

List of Tables

Table 1. Blueberry extract and powder confection formulations ...... 40 Table 2. Anthocyanin profiles of blueberry extract and whole lyophilized blueberry powder pre- confection processing ...... 50 Table 3. Blueberry extract and powder confection formulations ...... 72 Table 4. Demographic and compliance data of clinical cohort ...... 78 Table 5. Consumption frequency of blueberry confections ...... 83

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List of Figures

Figure 1. Base structure of flavylium cation ...... 8 Figure 2. Common aglycone anthocyanin families found in blueberries and their structures ...... 9 Figure 3. Pathways of biosynthesis of major blueberry anthocyanins11 ...... 11 Figure 4. Potential mechanisms of anthocyanin (Anth) absorption. SGLT, sodium– glucose co-transporter; Anth-3-gly, anthocyanin 3-glycoside; CBG, cytosolic b- glucosidase; LPH, lactate phlorizin hydrolase; UDP-GT, UDP-glucuronosyltransferase; Anth-gluc, anthocyanin42,46 ...... 15 Figure 5. Summary of hypothesized direct and indirect mechanisms of flavonoids and their metabolites on cognitive function improvement89,90 ...... 20 Figure 6. Main trends in the behavior of protein/polysaccharide mixtures128 ...... 28 Figure 7. A normal gelatin amino acid composition profile137 ...... 31 Figure 8. Sol- gel transition process of gelatin gels ...... 33 Figure 9. Representative HPLC spectra of BBEC and identification of each peak ...... 49 Figure 10. Anthocyanin retention of functional blueberry confections after manufacturing and 4- week storage at 4°C ...... 51 Figure 11. Percent freezable water calculated from DSC endotherms at approximately -20°C in blueberry extract and powder confections stored at 4°C for four weeks ...... 54 Figure 12. Rheograms of blueberry extract and powder confections over four week storage at 4°C. (A) Oscillatory strain sweep of BBEC at 25°C over four-week storage. (B) Oscillatory strain sweep of BBPC at 25°C over four-week storage ...... 57 Figure 13. Hardness of BBEC and BBPC over four- week storage ...... 59 Figure 14. Cohesiveness of BBEC and BBPC over four- week storage ...... 59 Figure 15. Gumminess of BBEC and BBPC over four- week storage ...... 60 Figure 16. Chewiness of BBEC and BBPC over four-week storage ...... 60 Figure 17. Sensory acceptability of blueberry confections...... 63 Figure 18. BBEC Just About Right analysis scores ...... 64 Figure 19. BBPC Just About Right analysis scores ...... 65 Figure 20. Clinical design ...... 73 Figure 21. Sensory acceptability of blueberry confections used in clinical trial ...... 80 Figure 22. BBEC Just About Right analysis scores ...... 81 Figure 23. BBPC Just About Right analysis scores ...... 82 Figure 24. Urinary recovery of BBEC and BBPC anthocyanins in 24 hour urine samples ...... 85 Figure 25. Total intake of food/ day ...... 128 xiii

Figure 26. Total calorie intake/ day ...... 128 Figure 27. Total fat intake/ day ...... 129 Figure 28. Total carbohydrate intake/ day ...... 129 Figure 29. Total protein intake/ day ...... 130 Figure 30. Total fiber intake/day ...... 130

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List of Equations

Equation 1. Percent freezable water calculation ...... 42 Equation 2. Retention of anthocyanins throughout blueberry confection manufacturing 45

Chapter 1. Introduction

1.1 Statement of Problem

78% of cancer patients who have received chemotherapy, and there are no current treatments available for the associated cognitive side effects which include memory loss, depression, and general feelings of mental “fogginess”.

Dietary interventions are a promising strategy to explore in order to mitigate the symptoms of chemo brain for multiple reasons. One of these reasons is that there are no current pharmacological treatments for CICI, and approval of such drugs is incredibly labor and resource intensive. Another reason is that there has been rich literature surrounding diet and how it can affect one’s health, specifically their cognitive health.

There has been particular success in the field of clinical dietary interventions with the use of functional confections with whole fruit inclusions, most likely due to their high consumer acceptance, shelf stability, and ability to modulate phytochemical release rate based on hydrocolloid selection and type of gel formed. However, even if functional

1 confections do present themselves as an adaptive delivery agent for a variety of phytochemicals there are many technical considerations to take into account when formulating a functional confection for use in a nutritional clinical trial, such as the compatibility of the bioactive-rich inclusion and its delivery matrix.

In functional food development, the choice of incorporating a bioactive-rich extract vs. a whole food containing bioactives remains a debatable issue. A direct comparison of consumer acceptability and product shelf stability is lacking for a specific food matrix containing either an extract or whole food. With growing pre-clinical and clinical data linking both whole blueberry and blueberry extract consumption with improved cognitive function most likely due to bioactive compounds in blueberries (mainly anthocyanins), the development of a fully characterized and stable delivery vehicle for blueberry bioactives is essential for use in future clinical studies looking at CICI.

This work serves as a preliminary examination of the differences in phyisco- chemical properties, consumer acceptance, and bioactive delivery seen when incorporating a whole lyophilized blueberry powder or an anthocyanin-rich blueberry extract into an equivalent gelatin confection matrix for future use in clinical studies looking at CICI, which has never before been formally studied.

1.2 Hypotheses

Both the blueberry extract and whole powder will produce consumer acceptable and shelf-stable delivery vehicles for blueberry bioactives, but their physical and chemical properties over one-month storage will vary. Also, we hypothesize that both

2 confections will be able to deliver equivalent amounts of anthocyanins to consumers after an acute dose, independent of bioactive source (extract vs. whole powder).

The following aims are proposed to investigate this hypothesis:

Aim 1: To investigate the effect of incorporating a whole lyophilized blueberry powder or a blueberry extract on a gelatin confection’s physico-chemical properties, consumer acceptance, and shelf stability over one month storage.

 Ascertain if a significant consumer acceptance exists between a functional gelatin

confection formulated with blueberry extract and whole blueberry powder

through sensory testing.

 Determine the effect of inclusion source (whole powder vs. extract) on bioactive

stability throughout processing and storage using high performance liquid

chromatography with photodiode array detector (HPLC-PDA)

 Demonstrate differences in water mobility of the blueberry powder and blueberry

extract confection using thermogravimetric analysis (TGA) and differential

scanning calorimetry (DSC).

 Elucidate variations in gel microstructure, strength, and texture of two functional

blueberry confections using rheological analysis and Instron.

Aim 2: Compare the two functional blueberry confections, one formulated with whole

fruit powder and the other with a phytochemical-rich extract, with regards to

bioactive delivery after consumption of an acute dose of blueberry anthocyanins.

 Compare urinary flavonoids composition of 24 hour urine after consumption of

one dose of each functional blueberry confection using liquid chromatography-

mass spectrometry (LC-MS)

Chapter 2. Literature review

2.1 Blueberries

Blueberries are a common indigo-hued North American fruit belonging to the

Vaccinium genus and have become of interest to the food industry due to their use in many different processing applications such as canning, freezing, freeze drying, puff- drying, as well as use in the fresh fruit market1. Blueberries belong to the larger woody shrub Ericacaea family which also includes berries such as huckleberries, cranberries, lingonberries, and bilberries2.

There are dozens of cultivars of blueberries grown in the U.S. today, falling under two main classifications, high-bush blueberries (Vaccinium corymbosum or “tall” blueberry plants), the hardier low-bush blueberry varietals (Vaccinium angustifolium or

“wild”), as well as rabbiteye cultivars (Vaccinium ashei) 3. High-bush blueberries are known for their tall plant heights as well as their abundant berry production and sweet flavor when compared to lowbush blueberries. Low- bush blueberries on the other hand, produce smaller fruits than high-bush plants, and are much more heterogeneous within the plant classification since many have been bred from wild ancestral clones. High-bush and low-bush blueberries differ not only in their physical phenotypes but also in their sugar and organic acid compositions. Low-bush blueberries contain both glucose and fructose, but no sucrose4,5, while high-bush varietals contain glucose, fructose, and sucrose6. With regards to organic acid profiles. High-bush blueberries have been found to contain high levels of citric acid (38-90% of total acids present), followed by succinic acid (approximately 17% of total acid profile)7. In contrast, low-bush blueberries are

5 characteristically dominated by malic, citric, and quinic acids. These differences in organic acid profiles found between high and lowbush blueberry varietals is thought to be a major driver of sensory differences among the two fruits3.

2.2 Commercial production and consumption of blueberries

2.2.1 U.S. Blueberry production

In 2011, the US grew 196,605 metric tons of blueberries in over 14 states, and total US consumption of blueberries has steadily increased since 1980. The United States is the top producers of blueberries globally, followed by Canada, Poland, and Mexico.

When looking at 2012 blueberry production information in the U.S. by state, Maine,

Michigan, and New Jersey are the top three producers for both fresh and processing blueberries, with total production ranging from 91.1 million pounds (Maine)-51.5 million pounds (New Jersey) per year. Michigan had the highest yield of blueberries/ harvested acre, producing 4,420 pounds/ acre harvested. Fresh blueberries were also sold for an average of $2.12/ pound after marketing prices were deducted that year, for a total of just

$800 million in sales in 20138.

2.2.2 U.S. Blueberry consumption

According to the USDA Economic Research Service (ERS) flavor, healthfulness, convenience, and year-round availability have contributed to increasing consumer demand for strawberries, blueberries, raspberries, and other berries, with per capita loss- adjusted availability growing from an average of 4.5 pounds per person per year during

1994-98 to 6.6 pounds during 2007-08 and to 9.9 pounds in 2014. Linking ERS’s loss- 6 adjusted food availability data with food intake surveys from 1994-2008 reveals that berries, like other fruit, are mainly consumed at home rather than away from home. The increase in berry consumption comes exclusively from purchases at grocery stores (the food-at-home market)9. Blueberries specifically are seeing the greatest amount of increase in their consumption compared to other fruits, with nearly 600% increase in per capita consumption from 1995-2015. Blueberries were also ranked the #1 preferred berries by consumers in a 2013 survey put out by the U.S. Highbush Blueberry Council

(USHBC), as well as being the only top ten consumed fruit with an expected increased consumption for 2017. This growing consumption of blueberries by the American public is also reflected by the increase in amount of commercial food products on the market containing blueberries, which has grown from 450 products in 1997, to more than 1,000 products in 2016 10. Outside of being consumed as fresh or frozen fruit, blueberries are commonly consumed in processed food products with the most popular products including preserves, syrups, fruit juices and beverages, and concentrates11.

2.3 Blueberry components and stability

2.3.1 Major blueberry components

Blueberries contain approximately 57 calories/ 100 g, they consist of 84.44% water, 0.74% protein, 0.33% lipids and 14.49% carbohydrates (9.96% sugars and 2.4% dietary fiber). Blueberries also contain 7 major minerals, including Ca, Fe, Mg, P, Na, and Zn, as well as multiple A, B, and D vitamins, and Vitamins E and K12. However, other than their rich vitamin and mineral content and overall low caloric burden per serving, blueberries are also rich in non- nutritive polyphenolic flavonoids, anthocyanins,

7 which impart their deep blue color, as well are the compounds commonly associated with the health benefits of consuming blueberries13.

2.3.2 Anthocyanin profile of blueberries

Anthocyanins are one of the most prevalent pigments seen in the plant kingdom, and are responsible for the red, blue and purple colors of many fruits and vegetables.

Anthocyanins are members of a larger family of phytochemicals known as flavonoids, and also a member of an even larger class chemical compounds known as polyphenols.14

While there are approximately 400 anthocyanins found in nature, the main structural differences among this vast array of compounds stems from differing numbers of hydroxyl groups, the nature and number of sugars attached to the base molecule, the position of the attachments, and the number of aromatic acids attached to the molecule.

The base structure of an anthocyanin, a flavylium salt, can be found in Figure 1.

Figure 1. Base structure of flavylium cation

Blueberries contain the most complex anthocyanin profile of commonly consumed berries, being comprised of approximately 20 different anthocyanins including three different glycosides (glucoside, galactoside, arabinoside) for each of five

8 major aglycone families (delphinidin, cyanidin, petunidin, malvidin, and peonidin) in addition to some acetylated forms in low concentrations (Figure 2)15. Malvidin usually predominates the anthocyanin profile of most blueberries, with the other four aglycone families having similar concentrations. Concentrations and numbers of anthocyanins present in blueberries can be greatly influenced by cultivar grown, growing environment, and stage of maturity. Total anthocyanin content in blueberries can range anywhere from

16 mg anthocyanins/ 100g fruit in the “Bright blue” cultivar from Georgia, USA to 823 mg anthocyanins/ 100 g fruit found in the “US-497” cultivar 16.

Figure 2. Common aglycone anthocyanin families found in blueberries and their structures

Anthocyanins are believed to be synthesized within the plant in order to protect itself from external damages such as photoinhibition, as well as an aid in pollination and plant seed dispersal11,17,18. Photoinhibition can be defined as the illumination of photosynthetic tissues in excess of the energy utilization potential of carbon reduction

9 which can lead to a marked decrease in photosynthetic capacity of the plant19. The production and distribution of anthocyanins within the plant are affected by both cultivar and environmental effects (season, temperature, soil composition, watering, and presence of pathogens), with cultivar being the dominant factor suggesting a high genotypic basis for anthocyanin production. This finding suggests that blueberries can be bred for maximized anthocyanin production and human health benefits. The pathways involved in anthocyanin biosynthesis in blueberries can be seen below in Figure 320.

Figure 3. Pathways of biosynthesis of major blueberry anthocyanins11

2.3.3 Stability of Anthocyanins to processing and storage

Most academic studies reporting on the health-promoting effects of blueberries have been carried out using fresh or freeze-dried berries, or an anthocyanin-rich extract isolated from fresh berries. Unfortunately, due to their perishable nature and limited seasonal availability, berries are commonly consumed in various processed forms

11 including juices, purees, jams, nectars, and canned products which warrants the study into the stability of berry bioactives in different food processes. These studies are particularly important in the case of anthocyanins since they have been shown to polymerize during both processing and storage, which could lead to both a decrease in product color as well as bioactivity13.

Brownmiller et. al performed a study detailing the change of anthocyanin composition with juice processing, canning, and pureeing when compared to fresh fruit.

Results showed that canning resulted in the greatest retention of anthocyanins when compared to purees and juices, likely due to the greater number of processing steps (i.e. pasteurization, freezing/thawing, concentration, etc.) that is involved in juice making. It is also thought that if one neglects to inactivate polyphenol oxidase before berry processing with a blanching step that could also be a major driver of anthocyanin degradation in blueberry products. Anthocyanins have been shown to degrade at temperatures above

70C, but losses can be mitigated if the thermal treatment is performed at lower temperatures (40-60C) for longer times21,22. When looking at anthocyanin composition changes during processing results showed delphinidin glycosides being the most unstable in processing because of its extensive hydroxylation on the B-ring (See below in Figure

3.), whereas malvidin seemed to be most stable due to its extensive methylation on the B- ring23. It is unclear whether it is anthocyanidin structure24 or the sugar the anthocyanidin was conjugated with25 that drove stability of the anthocyanin profiles through processing.

Some advanced processing methods such as radiant zone drying26, application of ultraviolet radiation type C27, pasteurization techniques, steam blanching28, high pressure

12 processing29, and application of modified atmosphere storage30 have been shown to be successful in anthocyanin preservation throughout processing and over shelf-life.

However, freezing and freeze-drying still remain the industry standard for long term storage of blueberries with the least amount of anthocyanin degradation.31

2.4 Absorption, metabolism and bioavailability of anthocyanins

2.4.1 Absorption of anthocyanins in humans

Consumption of anthocyanins has been linked to many health benefits in vitro in literature, however how these bioactive components are absorbed and metabolized in vivo is still largely unknown32–35. It has been historically thought that anthocyanins were poorly absorbed and metabolized by the body when compared to other flavonoids, such as quercetin, however new research suggests that this is not the case. Rather, it is currently thought that anthocyanin absorption has been greatly underestimated due to the oversight of many secondary metabolites present after anthocyanin consumption 36–39.

Anthocyanins are similar to other flavonoids since they are commonly found as glycosides in foods, which greatly influences their bioavailability40. Aglycone anthocyanins are primarily hydrophobic, and thus can passively diffuse through biological membranes, however glycosylation of the compound increases water solubility as well as limits passive diffusion and requires the use of a specific active transport mechanism or the cleavage of the -glycoside before anthocyanin absorption41,42. With the knowledge that the aglycone and glycoside anthocyanins are most likely absorbed very differently in the human body, there are several proposed mechanisms as to how the latter occurs. It is overall agreed upon in the scientific community that aglycone 13 anthocyanins are largely absorbed through passive diffusion in the small intestine, while the mechanism of how glycosylated anthocyanins are absorbed is more controversial.

One hypothesis states that anthocyanin glycosides are absorbed intact by way of a sodium-glucose cotransporter, while the second hypothesis suggests that glycosides are absorbed at the small intestine brush border via passive diffusion after the cleavage of the glycoside by lactate phlorizin hydrolase40,42,43. It is also likely that a portion of both glycosylated and aglycone anthocyanins will escape absorption in the small intestine and be left to be metabolized by gut microflora in the large intestine, where glycosides will be deglycosylated and aglycones will be subject to further metabolism41,42,44. Since both glycosides and aglycones have been isolated in plasma, it is likely that both absorption pathways of glycosides occur (Figure 4)45.

Figure 4. Potential mechanisms of anthocyanin (Anth) absorption. SGLT, sodium– glucose co-transporter; Anth-3-gly, anthocyanin 3-glycoside; CBG, cytosolic b- glucosidase; LPH, lactate phlorizin hydrolase; UDP-GT, UDP-glucuronosyltransferase;

Anth-gluc, anthocyanin42,46

2.4.2 Metabolism of anthocyanins in humans

Once being absorbed by the body, flavonoids are extensively broken down, supported by low levels of parent molecules being found excreted in urine after consumption47. The main reaction associated with flavonoid metabolism is glucuronide conjugation. The prevalence of this reaction stems from glucuronic acid high concentration being directly derived from glucose and glycogen and it’s the ability to conjugate with a wide variety of molecules48–52.Methylation and sulfation are also common reactions for anthocyanins to undergo when absorbed by the body, however they occur in different situations. Methylation is primarily driven by methyltransferases 15 and most commonly takes place within the liver46,50,53. Sulfation on the other hand, occurs mostly when low levels of phenolics are administered in the diet, and occurs by sulfotransferases which are distributed throughout the body in the cell cystol. It has been found that this sulfotransferases pathway is easily saturated, and thus is difficult to identify flavonoid sulfides in bio fluids such as blood and urine54,55. Overall, past studies investigating anthocyanin metabolism in humans have suggested that anthocyanins are not metabolized before entering circulation, however recent studies have found that this is most likely not the case. Recent detection of glucuronide, methyl, and sulfoconjugates of parent compounds has shown that between 68-80% of anthocyanins found in urine are actually metabolized derivatives36,39,56,57.

2.4.3 Pharmacokinetics of anthocyanins in humans

Complete pharmacokinetic data of anthocyanins is quite limited at this time.

From available data, it can be determined that max concentration of anthocyanins within the body is reached anywhere from 0.5-4 hours after the consumption of a 68-1300 mg dose. In past studies, the max concentration of anthocyanins in the body was within the range of 1.4-592 nmol/ L. For the parent glucosides, around 0.3-4% of the ingested dose have been detected in urine, and have been shown to have a half-life (t1/2) of 1.5- 3 hours in urine with max excretion occurring between 1-4 hours after ingestion41. Future work on anthocyanin pharmacokinetics should be focused on the role of colonic bacteria in metabolism and the metabolites they produce58–60, examining the full range of secondary metabolites produced by the consumption of anthocyanins61, as well as determining the bioactivity of all of these metabolites.

2.5 Anthocyanins and health

The health benefits associated with anthocyanins in past work include anticancer effects, anti-inflammatory effects, neuroprotective effects, anti-obesity and antidiabetic effects, and cardiovascular disease prevention62.

Anthocyanins have been shown to be successful against a wide array of cancers including metastatic melanoma63, colon cancer64, leukemia65, breast66, and others, though the majority of these studies being performed in cell culture and not in humans. Possible mechanisms of why anthocyanins possess anti-proliferative or anti-tumorigenic properties in cancer cell models is regulation of specific genes that are integral in the cellular signaling pathways that dictate tumor growth and cell apoptosis, such as the mitogen activated protein-kinase (MAPK)67,68. Anthocyanins have also been shown to activate nuclear factor kB (NF- kB), which are pathways that could be involved with cancer tumor growth and development69. While some of these pathways are ubiquitous to many cancer cells and it is clear anthocyanins are having some effect on some aspect of cancer gene expression, more work is required to elucidate how anthocyanins interact with specific cancer type metabolic processes.

Anthocyanins have also been associated with anti-inflammatory properties both epidemiologically and experimentally. Inflammatory responses are nonspecific tissue reactions to injury or infection that generally lead to tissue repair, however these inflammatory responses can also lead to the development of many human diseases when left unchecked70. Some examples of common illnesses thought to be caused by inflammation are colitis71, laryngopharyngeal reflux72, postprandial inflammation73, as 17 well as chronic pain74. Inflammatory responses are multi-step reactions that involve many downstream chemical cascades, and thus there are many opportunities for anthocyanins to mitigate an upregulated inflammatory response and aid in disease prevention. The enzyme Cyclooxygenase-2 (COX-2), nitric oxide synthase (iNOS), NF- kB cellular pathway, as well as cytokines (signaling compounds) have been shown in past works to be highly involved in promoting a body’s systemic inflammatory response75,76. Anthocyanins have been shown effective in downregulating COX-2, iNOS,

NF- kB, and cytokines across a variety of inflammatory disease models77,78, which reinforces anthocyanins as a promising adjuvant or preventative therapy for a variety of chronic diseases thought to be attributed to widespread systemic inflammation. However,

Graf et. al79 did find that a grape-bilberry anthocyanin rich juice did not provide any anti- inflammatory properties in systemic inflammation in rats, which also suggested that source, dose, and anthocyanin profile are pertinent factors in experiments examining anthocyanins and health, and need to be further studied.

Epidemiologic evidence demonstrates the link between those in the highest group of anthocyanin intake with lower relative risk of developing certain cardiovascular diseases, as well as a reduced incidence of cardiovascular disease mortality80,81.

Mechanisms supporting anthocyanins ability to mitigate inflammatory responses serve as credible hypothesis to these compounds’ protective role in the development of atherosclerotic lesions in a mouse model82. Anthocyanins were also shown to have the cardio protective effects by lowering oxidative modification of low-density lipoprotein

(oxLDL) through gene regulation83, increasing reverse cholesterol transport (RCT)84, 18 which is a process that entails the efflux of excess cholesterol from macrophages into the liver, as well as improvement of endothelial function85

2.6 Anthocyanins and neuroprotection

In addition to the health benefits discussed above, flavonoids such as anthocyanins also have rich research history with regard to increasing cognitive function, through both direct and indirect mechanisms which is summarized in Figure 5 below. It is also important to note that even though the bioavailability of anthocyanins seems to be quite low from past pharmacokinetic studies, these compounds and their metabolites have been shown to pass the blood brain barrier (BBB) and accumulate in an in vitro model of C6 glioma cells as well as multiple areas of the brain (hippocampus, cerebral cortex, cerebellum, ad striatum) pertinent to learning and memory function , though how exactly anthocyanins do this is still unkown86,87. It is also still unknown whether accumulation of flavonoids in brain tissues differs from a one-time acute feeding study of anthocyanins to a long-term feeding studies, which would have implications for how often one should consume flavonoid rich foods to achieve a clinically relevant increase in cognition88.

Figure 5. Summary of hypothesized direct and indirect mechanisms of flavonoids and their metabolites on cognitive function improvement89,90

Indirectly, anthocyanins have been shown to greatly regulate inflammatory processes, increase circulating nitic oxide (NO) species, and stimulate eNOS as well as improve overall vascular functions in vivo91. This specifically would help with cognitive function through the increase of cerebrovascular blood flow which could have many positive practical outcomes with regards to memory and learning. Cerebral blood flow rate has been shown previously to play a temporal and spatial role in neuronal activity and play an essential in brain activity and functionality92. The hypothesis is that

20 upregulated eNOS activity and elevated NO blood concentration ensures efficient vasodilation of the peripheral cerebral blood vessels in response to neural activity and thus improved practical function93. This hypothesis further reiterated by the observation that an NO deficiency and endothelium dysfunction has been associated with cognitive illness such as cerebral hypoperfusion (or ischemic stroke), vascular dementia, and

Alzheimer’s Disease.94

Hypotheses for the role of flavonoids in the direct improvement of cognitive function include modulation of genes that control synapse plasticity processes and relevant neuronal signaling pathways. Specifically it has been shown that flavonoid-rich blueberry supplementation of in aged rodents can activate ERK-CREB-BDNF and Akt- mTOR-Arc pathways, both cellular signaling pathways that are integral in neuronal growth and cell division, in the hippocampus95. Blueberry intake has also been shown to increase the level of NR2B subunits in the NMDA cellular receptors, which are cellular receptors that are important to directing synaptic plasticity and memory function in humans96. An increased level of NR2B subunits is specifically important in NMDA cellular receptors because they are thought to decrease with age, coinciding with a NR2A subunit increase, and are also associated with a greater degree of long-term potentiation (LTP) and stronger synaptic connections in the brain.97

This increase in cognitive function as a result of anthocyanin consumption, whether through direct or indirect mechanisms could result in improvement and protection of functional cognitive performance, memory performance, and motor performance. These observed neuroprotective properties of anthocyanin consumption

21 also indicates their potential application for the prevention of many neurodegenerative diseases such as Parkinson’s disease (PD), aging, and Alzheimer’s disease (AD).98 In

Parkinson’s disease, it thought that anthocyanins mainly express bioactivity indirectly by reducing the oxidative stress (OS) in glioma cells, which has a variety of positive implications for brain cell health such as increasing autophagy99, mitigating mitochondrial dysfunction, and reducing risk of ischemic stroke100. In addition to mitigating OS in brain tissues, anthocyanins have also been shown to directly induce

FK506 binding protein 52 (FKBP52) activation, leading to the reduction of hyper- phosphorylated Tau protein aggregation which is thought to be a major contributor to the development of AD101. It has also been hypothesized that anthocyanins may help delay the onset of AD indirectly as well through a second molecular mechanism, the decrease of amyloid-beta peptide (Ab) accumulation which is a known pathogenic feature of AD because of the redox imbalance it produces in the neuronal cells that ultimately leads to cell death102.

2.7 Blueberries and cognitive dysfunction

In addition to there being literature evidence supporting anthocyanins in general playing a neuroprotective role against cognitive disorders such as AD, ageing, and PD, there is also data linking blueberry and their anthocyanins specifically with increased cognitive function. Previous work explored the efficacy of blueberry extract in the mediation of symptoms of Alzheimer’s disease in a rodent model103. The rodents that were fed the NIH diet supplemented with blueberry extract for 8 months (4-12 months of age) performed better than control mice with regards to time it took to complete a Y

22 maze. However surprisingly, the blueberry supplementation did not have an effect on the pro-amyloid protein production in the brains of the mice, suggesting that cognitive ability can be improved via other mechanisms through diet intervention with blueberries independent of lessening amyloid load.

Blueberries have also been shown to improve cognition in rodent models of the less severe cognitive decline associated with ageing 104. Shukitt-Hale et al., explored various measures of cognition in aged rodents pre- and post an 8-week blueberry intervention or an 8-week strawberry intervention as well as chemical profiling of the lyophilized berry powders used for the diet supplementation of the controlled NIH-31 rodent diet. The measures of cognition tested included psychomotor behaviors such as rod walking, wire suspension, plank walking, inclined screen, and accelerating rotarod testing, all of which test the rat’s aptitude in movements associated with conscious mental activity. The Morris Water Maze test was also used to test the ability of the rodents’ working memory, as well as western blotting of insulin-like growth factor 1(IGF-1), which is a protein associated with neurogenesis and reversal of cognitive side effects of ageing. Results of the study showed that the aged rats fed the blueberry-supplemented diet showed improvements in cognition in many of these tests post-intervention compared to both the control and strawberry groups. IGF-1 levels were showed to increase in both berry groups of rats, however only the blueberry group showed a positive correlation between IGF-1 levels and working memory performance improvements in the

Morris Water Maze.

Blueberries and their specific bioactive compounds have been shown above to improve cognition in both Alzheimer’s and ageing rodent models105,106. Critically, there has also been recent evidence showing this cognitive improvement associated with blueberries also occurs in humans107. The experiment consisted of nine older adults with early memory changes who consumed wild blueberry juice daily for 12 weeks, and their pre- and post-cognitive abilities were assessed. The primary outcomes measured related to memory function, and included the Verbal Paired Associate Learning Test (V-PAL) and the California Verbal Learning Test (CVLT), both of which have been shown to be sensitive enough to elucidate age-related memory changes, mild cognitive impairment, and dementia108. Initial cognitive status of the cohort was recorded based on the Clinical

Dementia Rating (CDR) and all participants were classified in the mild cognitive impairment group before the start of the study. Results showed that blueberry juice participant scores improved significantly from baseline on both the V-PAL and CVLT tests, and also maintained significance in V-PAL but not CVLT improvement when compared to the placebo participants. Overall, this study demonstrated encouraging initial findings with regards to blueberries as a mitigation technique for human neurodegeneration.

2.8 Chemotherapy-induced cognitive dysfunction (CICI)

24 chemotherapy-induced cognitive dysfunction, CICI, also known colloquially in the medical community as “chemo brain”. Occurrences of chemo brain in cancer survivors is ubiquitous, with up to 70% of patients with cancer reporting that these cognitive difficulties persist well beyond the duration of their treatment, and for some, the impact it has on daily functioning is the most troublesome survivorship issue that they face. More alarming facts about CICI is that even though its incidence is so high among cancer patients, there is currently no accepted treatment or even therapy for the impairment, and until recent years chemo brain and its symptoms have been largely negated by the medical community110.

Studies have shown that in healthy rodents subjected to chemotherapy, major cognitive effects were observed when compared to baseline values. These include increases in cell death in the central nervous system, increase in oxidative stress, suppression of hippocampal neurogenesis, and decreases in levels of neurotrophic factors, which is hypothesized to be the cause of the cognitive symptoms of chemo brain111. Even though the biochemical markers of cognitive decline stated previously have been witnessed in animals, it has been difficult to translate these biological changes into measurable differences on current standardized neuropsychological assessments for people. Current cognitive function assessments are simply not sensitive enough to pick up the subtle changes experienced in CICI. Due to this difficulty, researchers are forced to rely on qualitative focus group data among cancer survivors to truly characterize the symptoms of CICI and begin to research ways to treat it.

2.9 Dietary interventions for cognitive improvement

The potential advantages of a food-based approach are as follows: (a) complex mixtures of bioactive phytochemicals impact multiple targets relating to beneficial health outcomes, (b) multiple components may have additive and synergistic activity112; (c) active agents, each provided at a lower dose, may reduce toxicity; (d) plant genetics and horticultural technology allow investigators to maximize phytochemical profiles of a plant for optimal nutrition; (e) modern food science technology and HPLC MS/MS provides a means to standardize food-based products for human studies; (f) food processing technology also provides a means to develop a consistent product of high bioavailability to specific tissues and enhance efficacy; and (g) products can be produced with excellent sensory characteristics and desirability in order to enhance adherence.

Previous work has demonstrated how a food-based clinical intervention has elicited high compliance in past clinical trials, in a variety of health states and using a variety of food products specifically formulated for bioactive delivery113–117. There has been particular success in the field of clinical dietary interventions with functional confections. There are numerous examples of confections in past works that were able to stably deliver clinically relevant levels of fruit bioactives over an extended period of

26 time114,118–120. This success of functional confections is most likely due to their high consumer compliance, shelf stability, and ability to modulate phytochemical release rate based on hydrocolloid selection and type of gel formed. However, even if food gels, specifically functional confections, do present themselves as an adaptive delivery agent for a variety of phytochemicals for the prevention or treatment of many disorders, there are many technical considerations to take into account when formulating a functional confection for use in a nutritional clinical trial.

2.10 Background of food gels

Gels can consist of a variety of materials including plant and animal tissues, polymers, and most relevant to this work, foods121. Also, the majority of foods consist of gels, and can produce many different textures depending on the choice of gelling agent, concentration used, and makeup of the food122. A gel can be described in many ways (1) a substance that exhibits solid-like behavior under mechanical forces123, (2) a solid that is self-supporting and can recover elastically after deformation124, and (3) polymeric molecules crosslinked to form a tangled interconnected network immersed in a liquid medium125. Gels can also be classified in a group known as “composite gels”, where one gel product is composed of two gelling agents, the most common of which is a combination of proteins (gelatin) and polysaccharides126. These composite gels present opportunities in food science, due to the range of interactions these protein and polysaccharide phases can have (ranging from no interaction, or “segregation”, to complete interaction, or “complexation”) which can be seen below in Figure 6 , and can have great consequences with regard to the resulting gel’s mechanical properties127. 27

Figure 6. Main trends in the behavior of protein/polysaccharide mixtures128

In this study, the interactions of the gelatin (protein) with the natural fruit pectin

(polysaccharide) that is present in lyophilized blueberry powder but not the polar blueberry ethanol extract will be explored.

2.11 Confectionary gels

Within the realm of food gels, there is an industrially relevant sub group, confectionary gels (CGs), whose market values has reached over $6 billion with continued annual growth129. CGs are characterized separately from other food gels by their high sugar contents as well as incorporation of a hydrocolloid or multiple hydrocolloids to provide varied textures130. Recent studies have demonstrated that the high sugar content present in CGs produce a very high solid gel network which is distinctly different from that of aqueous food gels, and thus further study is needed to fully characterize their physical, chemical, and mechanical properties131. The presence of 28 sugars affects the morphology of single biopolymer gels and the phase separation characteristics of a mixed biopolymer CGs. The main driver of CG texture is the hydrocolloid used for gelation, and even if sugars do not directly incorporate into the polymer network, they greatly contribute to CG behavior132. Although gelatin is the most popular gelling agent in CGs, other hydrocolloids can also be used such as agar, starch, pectin, alginates, and gums. Minor components of CGs include food acids, flavors, and colors133.

2.11.1 Role of water in confectionary gels

Water supports many chemical reactions within food systems, and thus removing it or binding it with salts or sugars will increase the shelf-life of a food product through the retardation of microbial growth, as well as slowing many degradative chemical reactions134. In addition to food safety and quality implications, water has significant effects with regards to food texture. Water will often act as a plasticizer in a CG system and allow for the hydrophilic portions of the gel system to become hydrated134. Water will also act a carrier solvent to dissolve the other CG components and aid in gel formation134. In a past study performed in a sucrose/starch/gelatin mixed matrix, it was shown that it was the gelatin that had the highest binding affinity for water among the tested components and therefore is inferred that it requires it to maintain gel structure134.

2.12 Gelatin

Gelatin’s primary role in food systems is as a gelling agent in order to bind water and provide texture, and is one of the most well studied biopolymers used in foods

29 today135. Specifically, gelatin is one of the leading hydrocolloids used in confectionary gels and candies and has the ability to successfully incorporate a variety of inclusions with high consumer acceptability, which makes it an excellent potential delivery vehicle for fruit bioactives in food-based nutritional interventions.

Gelatin is a thermoreversible gel formed in aqueous solvents through lowering the temperature, and is derived from collagen via controlled acid or alkaline hydrolysis136.

The collagen used to produce gelatin can be derived from bovine (cow), porcine (pig), or piscine (fish) origins, and thus gelatin is not considered a vegan or kosher food product137

The functional properties of gelatin can be influenced by two main factors: the collagen source and the extraction methodology used134.

2.12.1 Gelatin composition

The typical composition of gelatin is 14% moisture, 84% protein and 2% ash138.

The protein portion of gelatin is usually comprised of the following amino acids: glycine, proline and hydroxyproline and their proportions can be seen below in Figure 7137.

Figure 7. A normal gelatin amino acid composition profile137

These amino acids reorganize themselves into long chains that then interact and entangle further forming the overall gelatin network. Gelatin chains commonly are made up of glycine –X-Y repeating networks, where X and Y are typically proline or hydroxyproline, with glycine providing chain flexibility139. It is these repeating sequences that lead to the formation of triple helices within the gelatin network, similar to many other protein networks that are stabilized by hydrogen bonding and ultimately immobilize water in the system. These triple helices than interact with each other via secondary forces to create the final three-dimensional network 136,140.

2.12.2 Gelatin gelation

Gelatin can transition from its soluble state to its gel state (the sol-gel transition) at protein concentrations anywhere above 2-3%141. There are two different ways in which a gelatin solution will undergo this sol-gel transition, one which is through a direct 31 solution and the other is through an indirect solution. A direct solution, the most common method, is created when gelatin is added to heated water (60-80°C) with high speed agitation to prevent clumping137. An indirect solution on the other hand is the more time intensive method and is produced when gelatin granules are allowed to soak in cold water for some allotted time period until the granules swell. Then, the resulting mass is heated to 50-60°C until full dissolution is reached137.

There are several theories behind the mechanism of gelatin CGs, with many factors affecting which one is utilized in specific gelatin events such as type of gelatin, concentration of gelatin and temperature history of the solution. When a solution containing gelatin is heated above 40°C, the hydrocolloid molecules begin to act as a traditional synthetic polymer, reorganizing themselves into individual macromolecules

(average molecular weight of 2x105 Daltons) with random coil configurations. These random coils are made up of single polypeptide chains at this point, and can still be untangled, however once the mixture is cooled, these single peptide chains restructure themselves into triple helices which leads to the sol-gel transition and eventually full gelation137,142.

The gelatin process can be spilt into two major segments: setting and ageing.

Setting involves the orienting of the repeating glycine –X-Y repeating networks so that the three-dimensional gelatin network is established142. Ageing involves both the adjustment of the gelatin network through both hydrogen bonding and moving of the triple helices. The initial interchain links that were formed via the setting stage, are strengthened as the environment approaches isothermal conditions, as only the best-

32 established links can be maintained at a single temperature. Since gelatin networks typically form rather slowly, problems from rapid hydrocolloid aggregation, such as brittle gels, are rarely encountered with gelatin140. A visual depiction of the setting and ageing process in the formation of gelatin gels can be seen below in Figure 8, which was adapted from the review of confectionary gels by Burey et. al.143

Figure 8. Sol- gel transition process of gelatin gels

There are numerous factors that affect the gelatin gelling process. Some of these factors include temperature, water concentration, presence of other ingredients in the gel, pH, and sugar concentration. With regards to temperature, it is known that the lower the ageing temperature is, the quicker the helix formations, but also the less stable they will be144,145. Also, small increases in temperature will result in a melting of some established junctions, and even larger increases in temperature could lead to complete reversal of gelatin because gelatin is a thermoreversible gel145. Water is essential in order for the gelatin gel to form, but the lower the water levels present and the higher the gelatin concentration in the system affects the final gel by speeding up the time it takes to gel. 33

The change in modulus occurs at increasingly steeper rates with increasing gelatin concentrations. Also, it has been that shear applied to the gelatin system will also have implications on the viscosity of the sol-gel transition144,146. The presence of other ingredients in a gelatin system, if in high enough concentrations, also can have effects on gelation and final gel properties. In the case of starches, the amylose molecules can leach into the gelatin phase and interfere with triple helix cross-linking and thus may weaken the resulting gel147. The pH of the gelatin system will affect the viscosity of the sol-gel transition as well as the final gel turbidity. Due to the fact that acid has been shown to degrade a gelatin network, food acids are usually one of the last ingredients to be added to gelatin CGs137,138. Finally, sugar can also affect the final gelatin network. In addition to stabilizing the gel configure, sugar also aids in the gelatin dissolution by creating a continuous liquid phase with the gelatin131. However, above a certain point sugar will destabilize the final gel matrix by binding too much of the water in the system, rendering it unavailable for gelation, which can be a concern using gelatin in CG applications131,148.

Chapter 3. Comparison of consumer acceptance and physico-chemical properties of blueberry extract and whole blueberry powder confections designed for a future clinical trial

3.1 Introduction

Advances in chemotherapy and other cancer treatments have greatly increased the rate of survival among many of the most common cancers, but these improvements in cancer treatment have also caused new side effects in patients that greatly affect their daily lives. One of the most common of these secondary side effects is chemotherapy- induced cognitive impairment (CICI) also known colloquially in the medical community as “chemo brain”. Chemo brain can be described as a state of prolonged cognitive dysfunction resulting from administration of a variety of common cytotoxic chemotherapeutic agents, including doxorubicin and cyclophosphamide149, which may last years after cessation of chemotherapy150,151. Cognitive occurrences of chemo brain in cancer survivors is ubiquitous, with up to 70% of patients with cancer reporting that these cognitive difficulties persist well beyond the duration of their treatment, and for some, the impact it has on daily function is the most troublesome survivorship issue they face111. Studies have shown that in healthy rodents given chemotherapy, major cognitive effects are observed when compared to baseline values. These include increases in cell death in the central nervous system, increase in oxidative stress, suppression of hippocampal neurogenesis, and decreases in levels of neurotrophic factors, and are hypothesized to be possible causes of the cognitive symptoms of chemo brain111. Of great concern is that even though its incidence is so high among cancer patients, there is currently no accepted treatment or even therapy for this impairment.

The consumption of a diet high in anthocyanins and other flavonoids has been implicated with numerous health benefits such as obesity control, diabetes control152, cardiovascular disease (CVD) prevention153, cancer prevention154, and most notably in this context, improvement of visual and brain function155. Blueberries present themselves a promising candidate for nutritional interventions looking at their effect on cognitive dysfunction due to their high anthocyanin content (`129.2 mg anthocyanins/ 100 g fruit156), high consumer acceptance, regular domestic availability, and ample pre-clinical and clinical literature evidence, in particular, in the area of cognitive dysfunction.

Consumption or treatment with blueberries, blueberry juice, or blueberry extracts have demonstrated potent cognitive improvement in in vitro cell culture studies157, in vivo rodent models103,104,106,158, and in human clinical studies107,108,159. Although the relationship between blueberries and cognitive health has been suggested in past literature, the intervention agents used in these studies have not been well-characterized nor optimized for the delivery of bioactives. Therefore, a need exists to characterize a deliberately formulated food rich in blueberry anthocyanins that meets consumer acceptability requirements.

Though it is well recognized that what people eat has a drastic effect on their health, in functional food development, the choice of incorporating a bioactive-rich extract vs. a whole food containing bioactives remains a debatable issue. Historically, researchers have favored a reductionist approach and the use of extracts and/or specific bioactive compounds when looking at prospective efficacy against specific diseases.

However, the potential advantages of a food-based approach include: complex mixtures

36 of bioactive phytochemicals impact multiple targets relating to beneficial health outcomes160, multiple components may have additive and synergistic activity; active agents, each provided at a lower dose, may reduce toxicity; food processing technology also provides a means to develop a consistent product of high bioavailability; and products can be produced with excellent sensory characteristics and desirability in order to enhance adherence. And, even though this debate persists in the nutrition community between incorporating a whole food vs. an extract into functional food matrices, a direct comparison of bioactive uptake, consumer acceptability, and product shelf stability is lacking for a specific food matrix containing an extract or whole food in current literature.

Gelatin is a thermoreversible gel formed in aqueous solvents through lowering the temperature, and is derived from collagen via controlled acid or alkaline hydrolysis136.

Gelatin’s primary role in food systems is as a gelling agent in order to bind water and provide texture, and is one of the most well studied biopolymers used in foods today135.

Specifically, gelatin is one of the leading hydrocolloids used in confectionary gels and candies, and has the ability to successfully incorporate a variety of inclusions with high consumer acceptability, which makes it an excellent potential delivery vehicle for fruit bioactives in food-based nutritional interventions. Historically, our lab has successfully used several confection matrices as delivery vehicles for bioactive food components in clinical trials with high consumer acceptability, phytochemical stability, and compliance118,119,161. This, in addition to the ability to manipulate confection’s gelation mechanism and structure via hydrocolloid selection161 and large market share populated

37 by confections in today’s economy129 make confections an excellent strategy to develop high impact and translatable functional food products.

Therefore, the objectives of this study were to 1). Develop and characterize two blueberry functional gelatin confections, one formulated with a blueberry extract (BBEC) and one with whole lyophilized blueberry powder (BBPC), for future clinical trials and determine their sensory acceptability, and 2). Compare the physical and chemical properties of a gelatin confection matrix containing either a blueberry extract or whole fruit powder over one month storage. We hypothesize that both the blueberry extract and whole powder confections will produce consumer acceptable and shelf-stable delivery vehicles for blueberry bioactives yet the physical properties of these confections will differ.

3.2 Materials and Methods

3.2.1 Blueberry confection preparation

The whole blueberry powder (N1112 Blueberry Powder) and blueberry extract

(N1077 VitaBlue) used as inclusions in the gelatin confections were made from the same raw materials (Vaccinium corymbosum) and purchased from the same supplier

(Futureceuticals, Momence, IL). Both confections were prepared by mixing gelatin

(Knox Gelatin, Treehouse Foods, Inc., Oakbrook, IL), sugar (Domino Foods Inc., Iselin,

NJ), citric acid (Tate and Lyle, Decatur, IL), Jell-O Berry Blue (Kraft Foods, Northfield,

IL), and either the blueberry extract or powder described previously. This mixture was then stirred and heated on a hot-plate until reaching 100C, which typically took approximately 20 minutes. Upon reaching boiling, the confection base was removed from 38 heat and moved to a 9X9 in square baking pan lined with parchment paper and moved into a refrigerator (4 C) to solidify, which took approximately 3 hours. Confections were then removed from the pan and cut into squares (1in x1in x0.5 in), placed in Ziploc bags, and stored at 4 C, avoiding air and light, to mimic how the confections would be stored if used in a clinical trial. The final formulation the blueberry extract and blueberry powder confections can be seen below in Table 1. Both confections were formulated to deliver approximately 320 mg of anthocyanins/ 100 g (the extract was found to be ten times as concentrated as the whole food powder with regards to anthocyanin content), or about ten confections, which is equivalent to eating 2 cups of fresh blueberries12.

Table 1. Blueberry extract and powder confection formulations

Blueberry Extract Blueberry Powder Ingredient Confection (%) Confection (%)

Water 37.0 37.0

Sugar 29.3 2.0

Jello Berry Blue 26.0 26.0

Knox gelatin 4.5 4.5

Citric acid 0.5 0.5

Blueberry Extract 2.8 0.0

Blueberry Powder 0.0 30.0

total 100.0 100.0

Time- dependent changes in the confections were determined from samples stored for 0h, 24 h, and in samples after storage (7,14, 21, and 28 days) at refrigeration temperature with relative humidity range from 38% to 42%.

3.2.2 Thermogravimetric Analysis (TGA)

Thermogravimetric analysis was used to analyze the total amount of moisture present in each blueberry confection over one month storage, as well as elucidate whether there were different species of water present in functional gelatin confections made with a blueberry extract verses a whole blueberry lyophilized powder. This was calculated as a

40 result of mass loss of each confection with heating at Day 0, Day 1, and then Weeks 1-4.

A TGA 550 (TA Instruments, New Castle, DE) was used to analyze 10-20 mg samples of confections at a heating rate of 5°C/min from 20 to 200°C. Samples were placed in hermetically sealed aluminum pans. TA TRIOS Software (TA Instruments, New Castle,

DE) was used to compute the first derivative of the weight loss curve, as well as calculate

% moisture content of the sample. Three replicates from two batches of each confection type were analyzed to gauge batch-to-batch manufacturing variability. The percentage of mass loss of the confection sample up to 160°C was analyzed and assumed to be from vaporization of water162. Only one peak of accelerated mas loss was observed in the first derivative weight loss curve before 160°C, and thus it was assumed only one species of water was present in both confections. Loss of mass past 160°C was assumed to be degradation of the sample from heating.

3.2.3 Differential Scanning Calorimetry (DSC)

Thermal analysis was performed using differential scanning calorimetry using a

DSC 2500 (TA Instruments, New Castle, DE) equipped with a cooling system. A hermetically sealed aluminum pan was filled with 10-20 mg of confection sample. An empty pan was used as a reference and nitrogen was used as the purge gas. For analysis, sample temperature was lowered to -50°C, held isothermally for 3 min, and then heated to 100°C at a rate of 5°C/ min, adapted from a method used previously163. Thermal transitions observed in the resulting DSC thermograms were integrated using TA TRIOS software (TA Instruments, New Castle, DE). Endothermic peaks seen in ranges from -

40°C to 10°C were attributed to the melting of ice and used to calculate the percent freezable and unfreezable water seen in the confections using the Equation 1 below164:

Equation 1. Percent freezable water calculation

%FW = Peak Enthalpy x 1 x 100 Latent Heat of Fusion of Ice weight of water/weight of sample as cited in past literature in high sugar matrices 161. Like with the TGA analysis, measurements were performed on three replicate samples from two separate batches of each confection type on Day 0, 1, and Weeks 1-4.

3.2.4 Texture Profile Analysis

An Instron 5542 (Instron Corporation, Norwood, MA) affixed with a 35 mm steel parallel plate compressed confection samples (approximately 1 cm length x 1.0 cm height x 1.0 cm diameter) with a crosshead speed of 3 mm/s to 30% deformation with two uniaxial compressions fitted with 100 N cell load. Hardness (N), cohesion energy, gumminess (N), and chewiness (N) were determined using Blue Hill software (Instron

Corp., Norwood, MA) and described in detail by Bourne165. Ten replicates were performed for each batch of each confection because large variations between samples were observed and all samples were allowed to reach room temperature (~ 3 hours) before the analysis took place.

3.2.5 Rheological Analysis

Rheological testing of blueberry confections was conducted with a Discovery HR-

2 Rheometer (TA Instruments, New Castle, DE) affixed with a 20-mm steel parallel plate and a 2.0 mm gap. To obtain information regarding the viscoelastic behavior of the confections under applied stress, confection samples that were centrally removed (1 cm 42 height x I cm length x 2 mm thickness) were used for oscillatory strain sweeps in the viscoelastic range (10-2-104 %). The viscous modulus (G’), elastic modulus (G”) were monitored throughout the analysis using the TA TRIOS Software (TA Instruments, New

Castle, DE). Oscillatory analyses were performed in triplicate for each confection batch, and all samples were allowed to come to room temperature (~ 3 hours) before the analysis took place.

3.2.6 Bioactive Identification and retention

Both blueberry confections were analyzed using high performance liquid chromatography with a photodiode array detector (HPLC-PDA) to quantify anthocyanin concentration in both the blueberry extract and blueberry powder before confection preparation, as well as bioactive retention in the blueberry extract and powder confections after manufacturing, and throughout 4 weeks of storage. Identification of which anthocyanins were present in the confections was also performed using UHPLC with a mass spectrometer (MS) detector.

Extraction of powders and confections for analysis

The blueberry extract and powder were extracted for HPLC analysis using the following procedure. 100 mg of powder was weighed and added to 2 mL of 70:30:1

Methanol (MeOH): water (H2O):Trifluoroacetic acid (TFA) extraction solvent. The suspension was then vortexed for one minute, placed in a bath sonicator for five minutes

(FS3OH Sonicator, Fisher Scientific, Fair Lawn, NJ), and centrifuged at 4000 rpm for five minutes with a IEC HN-SII centrifuge (Damon Corp., Needham Heights, MA). The resulting supernatant was removed to a clean 11 mL glass vial (Fisher Scientific,

Pittsburgh, PA) with a disposable glass pipet (Fisher Scientific, Pittsburgh, PA, USA).

Solvent of MeOH: H2O: TFA was used to extract the pellet twice more, until supernatant was mostly colorless. Extraction of the confections were equivalent to the previous procedure with the following exceptions. Approximately three grams of confection were frozen with liquid nitrogen, and then pulverized with a mortar and pestle into a powder.

One gram of this powder was then added to 3 mL of extraction solvent and the procedure was repeated. All extracts were then filtered through PTFE filters (Fisher Scientific,

Pittsburgh, PA; 0.2 μm, 13 mm diameter) before analysis.

Anthocyanin Identification

An Agilent Infinity 1290 UHPLC with photodiode array detector combined with an Agilent 6495 QqQ (Agilent, Santa Clara, CA) was used for the identification of anthocyanins. Separation was carried out on a Symmetry C18 (75 mm × 4.6 mm i.d, 3.5

μm particle size) reversed phase column (Waters). The mobile phase for separation consisted of 10% (v/v) formic acid in water (A) and acetonitrile (B). Initial mobile phase composition, 100% A and 0% B, was followed by a linear gradient to linear gradient from 0% B to 15% B, 0-15 minutes; linear gradient from 15% B to 20% B, 15–17min; linear gradient from 20% B to 95% B, 17–17.5 min; isocratic elution 95% B, 17.5–18 min; linear gradient from 95% B to 0% B, 18-18.1 min, isocratic elution 0% B, 18.1–

19.5min, flow rate 1.3mL/min; injection volume 0.3 µL for BBE and BBP. The MS analysis was performed in positive ion mode with electrospray ionization and SRM transitions for each anthocyanin. IDs were confirmed by accurate mass and fragmentation

44 on an Agilent 6550 QTof and supported by literature evidence for blueberry anthocyanins.

Quantification

After anthocyanin identification, the blueberry extract, powder, and confections were analyzed by HPLC to quantify total anthocyanins present and to determine the changes of blueberry bioactives during confection production and subsequent storage.

Quantification in all blueberry samples was carried out using an Agilent 1100 series

(Agilent, Waldbronn, Germany): G 1322A degasser, G 1328A manual injector, G 1311A quaternary pump, and G 1365A MWD controlled by the ChemStation software (Agilent).

Chromatographic separation was performed with the same column and method as those in the identification step. Extract samples were injected with a volume of 1 μL, while blueberry powder and both confection samples were injected with a volume of 5 μL.

Absorbance at 520 nm wavelength was recorded. Anthocyanins were quantified with cyanidin 3-glucoside as external calibrant13 with concentrations ranging from 2 to 264

μmols/L (R2=1).

Retention of anthocyanins in blueberry confections.

The retention rate of the anthocyanins through confection preparation and storage was calculated using the below formula,Equation 2. Retention of anthocyanins throughout blueberry confection manufacturing Equation 2 :

Equation 2. Retention of anthocyanins throughout blueberry confection manufacturing

% Retention = C product x 100 C BBE/BBP added x C anthocyanins in raw material 45 where Cproduct = concentration of anthocyanins in products post-processing or storage,

CBBE/BBP= amount of blueberry extract or powder added to the confection (g) , and

Canthocyanins= amount of anthocyanins in blueberry extract or powder pre-processing and storage (mg/g). Cproduct and Canthocyanins were obtained from HPLC quantification of extract, powder, and confections, and CBBE/BBP added were obtained from the confection formulation.

3.2.7 Sensory analysis of blueberry confections

Sensory evaluation of the two blueberry confection formulations was used to optimize confections for physicochemical analysis and storage studies, as well as to determine if there was a significant consumer preference. Once OSU Institutional Review Board approved (OSUIRB # 2017E0569) this research with a category 6 (NIH criteria) exemption, participants (n = 75) 18 and older were recruited. Eligible participants were not allergic to blueberries, pectin, gelatin, artificial sweeteners, artificial flavorings as well as wheat or Gluten-free crackers; not diabetic or without altered glucose metabolism; and also had no visual or sensory impairments. If all inclusion criteria were met, participants were allowed to evaluate the blueberry confections (approximately 10 g sample held in 2 oz, sealed soufflé cups). Ambient fluorescent light and overhead incandescent light illuminated each sensory booth. Using a 9-point hedonic scale (1 = dislike extremely to 9 = like extremely) consumers were asked to evaluate the acceptability (overall, aroma, flavor, sweetness, texture, and grittiness). Evaluations of confections were conducted in the OSU sensory facility designed according to ISO

Standards (2007)166. Hence consumer testing was conducted using individual booths

46 equipped with pass through windows and facility environment was controlled for temperature, air flow, and noise. Ambient fluorescent light and overhead incandescent light illuminated each sensory booth. Additionally, a 5-point Just About Right (JAR) scale (1 = much too weak to 5 = much too strong) assessed the appropriateness of the intensity of fruit aroma, stickiness, sweetness, sourness, bitterness, and fruit flavor.

Finally, the panelists were also asked to compete a paired preference test in which at the end of the sensory evaluation they answered which of the two confections they preferred.

Results of the sensory evaluation were recorded using a Compusense software (Guelph,

Ontario,CAN).

3.2.8 Statistical analysis

Mean± SD were reported and were performed with SPSS statistical software (IBM, North

Castle, NY). Paired preference test results of the two confections were analyzed using binomial statistic tables167. JAR results were displayed as histograms and attributes were considered unsatisfactory if less than 70% of respondents rated it as “Just About Right”.

Paired t-tests were used to compare mean acceptability scores of the confections.

Unpaired t-tests were used to compare the shelf study parameters measured from both confections at individual time points: anthocyanin content, total moisture content, % freezable water, texture parameters, and rheological properties. One-way ANOVAs were used to compare shelf-study parameters within confection type across the six measured time points. A p≤ 0.05 was considered to be significantly different.

3.3 Results and Discussion

3.3.1 Anthocyanin identification, quantification, and retention after confection manufacturing and 4-week storage

Anthocyanins present in both the blueberry powder and extract as well as the final blueberry powder and extract confections were identified and quantified with a combination of UHPLC-MS/MS, external standards, UV-Vis, and reported accurate masses of anthocyanins in literature and were similar to those found previously16,168. The blueberry extract and lyophilized powder were made commercially from the same raw material (Vaccinium corymbosum), and anthocyanin profiles in both powders and both confection types after manufacturing were found to be equivalent (a representative chromatogram of the blueberry confections can be seen below in Figure 9.

Figure 9. Representative HPLC spectra of BBEC and identification of each peak a Abbreviations of tentative IDs are as follows: Dp: delphinidin, Cy: cyanidin, Pt: petunidin, Peo: peonidin, Mal: malvidin, gal: galactoside, glu: glucoside, ara: arabinoside

The blueberry extract powder was approximately ten times as concentrated

(115.29±0.43 mg anthocyanins/ g powder) as the whole blueberry powder (10.83±0.53 mg anthocyanins/ g powder), which agrees with levels that have been seen in dried high bush blueberries169. This difference was taken into account when formulating the confections by incorporating 30.0 grams of whole blueberry powder compared to 2.8 grams of the extract per 100 g of confection for a total of approximately 320 mg anthocyanins/ 100 g confections (~5 confections). The effect of confection manufacturing on anthocyanin levels was investigated. Two petunidin compounds, the aglycone as well as an acetylated species, that were present in both the blueberry extract as well as the 49 whole fruit powder pre-processing, were not seen after confection manufacturing, most likely due to low initial concentrations and thermal degradation170. The anthocyanin profiles present in both the blueberry extract and blueberry powder are shown in Table 2.

Table 2. Anthocyanin profiles of blueberry extract and whole lyophilized blueberry powder pre- confection processing.

Tentative anthocyanin Concentration (mg anthocyanin / g powder) identification Blueberry extract Blueberry powder Delphinidin-3-galactose 18.27±0.05 1.99±0.12 Delphinidin-3-glucose 7.25±0.03 0.78±0.05 Delphinidin-3-arabinose 9.51±0.05 1.14±0.06 Cyanidin-3-galactose 2.22±0.02 0.24±0.01 Cyanidin-3-glucose 1.24±0.01 0.08±0.01 Petunidin-3-galactose 11.13±0.02 1.00±0.05 Petunidin-3-glucose 7.06±0.03 0.61±0.03 Petunidin-3-arabinose 4.00±0.02 0.39±0.02 Petunidina 0.18±0.01 ---b Petunidin-6-acetyl-3- 0.70±0.04 0.05±0.00 hexosidea Peonidin-3-galactose 0.28±0.01 0.03±0.00 Peonidin-3-glucose 0.44±0.01 0.02±0.00 Malvidin-3-galactose 27.71±0.09 2.23±0.10 Malvidin-3-glucose 15.03±0.06 1.25±0.05 Malvidin-3-arabinose 10.28±0.04 1.03±1.03 Total anthocyanins 115.29±0.50 10.83±0.73 aAnthocyanins lost in confection manufacturing

50 bNot detected

Using Equation 2 for bioactive retention, it was seen that at immediately after confection manufacturing (Day 0), the BBEC retained 65% of its anthocyanins (2.09 mg anthocyanins/ g confection, or 209 mg anthocyanins/ 100g) and BBPC retained 87% of its theoretical anthocyanins (2.83 mg anthocyanins/ g confection, or 283 mg anthocyanins/100 g batch) (Figure 10).

3.5 * * 3 * * * * 2.5

1.5

ACN (mg/g (mg/g ACN confection) 0.5

0 Day 0 Day 1 WEEK 1 WEEK 2 WEEK 3 WEEK 4 TIIME

BBEC BBPC

Figure 10. Anthocyanin retention of functional blueberry confections after manufacturing and 4- week storage at 4°C

At week 4, anthocyanin retentions were slightly higher then values measured at

Day 0, with results showing BBEC had retained 72% and BBPC had retained 96% of initial anthocyanins. Though the anthocyanin content of the BBPC was statistically higher across all six time points (p-values Day 0:≤0.001, Day 1: ≤0.001, Week 1: ≤0.001,

Week2: 0.020, Week 3: ≤0.001, and Week 4: ≤0.001), there was no statistically significant difference in anthocyanin content/ g confection found within confection type 51 across time indicating bioactive stability of the functional gelatin delivery matrix with regards to blueberry anthocyanins. This finding is contrary to past literature that showed up to 50% degradation of berry anthocyanins at thermal processing temperatures of 70°C.

Higher retention in the blueberry confections may be due to removal from heat at the moment the slurry reached 100°C along with the storage at 4°C which greatly slowed the rate of degradation170. Bioactives in both confection types remained relatively stable over the four-week storage, yet the blueberry powder confection retained a significantly higher percentage of its overall anthocyanin content. It is unclear why the stability of anthocyanins from whole-foods, such as the blueberry powder confection, retained more of its overall anthocyanin content when compared to a food-derived extract source, such as the blueberry extract confection, after undergoing identical thermal processing and warrants further research to add to the foods for health scientific debate171. However, a hypothesis as to why this occurred can be made surrounding the difference in sugar concentrations between the two formulations. It has been demonstrated in previous work that an increased °Brix will also increase the rate of anthocyanin degradation during heating above 70°C172. A main difference between BBEC and BBPC was that the bulk of the blueberry extract confection formula was added sucrose. Thus it is reasonable to hypothesize that the higher degree of bioactive degradation observed in this study could be attributed to the BBEC’s higher product sugar content (increasing solid content) leading to increased reaction rate due to a concentration effect 173.

3.3.2 Differences in water-binding of blueberry extract and lyophilized blueberry powder confections

Though often overlooked, water has been considered the most important functional ingredient in confectionary science with regards to confection texture, physical properties, quality, and shelf- stability174. Characterizing water state and distribution in confections can help determine final quality and stability of the products174. Water that is unable to be frozen at sub-freezing temperatures, unfreezable water (%UFW), has been used as an indicator of the stability of a food175. TGA results showed that total moisture levels of both the blueberry extract (30%) and blueberry powder confection (35%) were considerably below that of the levels of formula water added (37%) due to evaporation that occurred during confection processing, but remained stable at 30% for the blueberry extract and 35% for the blueberry powder confection over the four weeks of storage (data not shown), which is typical for gummy candies176.

However, even though overall moisture content was similar in the two confection formulations, a significant difference was found in the amount of freezable water(%FW), with the blueberry powder confections containing significantly more freezable water

(FW) (ranging from approximately 0.7- 1.75% FW) than those made with blueberry extract (≤0.04% FW after normalizing for differences in overall moisture content) across five of the six time points (p-values Day 0:0.016, Day 1: 0.016, Week 1: 0.003, Week 3:

≤0.001, and Week 4: 0.017). No significant differences were seen in freezable water content in a single confection type across storage time and distribution of %FW contents,

53 as well as representative thermograms for both the BBEC and BBPC can be seen below in Figure 11.

BBE C 20 * * * 15 *

BBP % FW % 10 C *

0 Day 0 Day 1 WEEK1 WEEK 2 WEEK 3 WEEK 4 Day 0 Day 1 WEEK1 WEEK 2 WEEK 3 WEEK 4 BB Extract (BBE) BB Powder (BBP) Time

Figure 11. Percent freezable water calculated from DSC endotherms at approximately

-20°C in blueberry extract and powder confections stored at 4°C for four weeks

Such differences are likely due to the greater binding capacity of sucrose177, the major bulking agent found in the extract confection formulation, in comparison to fruit pectin, glucose, and fructose, which are the major water-binding agents found in the blueberry powder confection formulation. Previous work has shown that the molecular weight of a food ingredient can have direct correlation to its water activity lowering or water binding capacity, with a lower molecular weight producing a lower water activity174. This corresponds with what was found in this study, with sucrose (342.3 54 g/mol) having a lower molecular weight than fruit pectin (60,000-130,000 g/mol178), and also a better water binding capacity observed in the DSC results in Figure 4. In addition to the molecular weight contributions to the increased water binding by sucrose, there are also entropy considerations that result in preferential water binding by sucrose when compared to other components in the system. In a hydrocolloid mixture with sugar, such as the BBEC and BBPC, sucrose will outcompete other solutes in the system for water, due to the resulting hydrogen bonds between water molecules and the sucrose hydroxyl groups that are entropically favorable177. There is also an entropy cost associated with the mixing and incompatibility of rigid linear molecules (fruit pectin) with the protein phase (gelatin) of the gel in the whole fruit powder blueberry confection which could also attribute to the lesser water binding seen in BBPC126,179.

There was also a much greater degree of variance in the % FW results seen in the blueberry powder confection when compared to the blueberry extract confection likely due to the heterogeneous nature of the whole blueberry powder (including lipophilic and complex carbohydrate fractions12 that were removed in the ethanol extraction used to obtain the blueberry extract powder) which could cause complex interactions with water in the gel system. Additionally, freeze-dried model fruit powders were shown to absorb more water compared to other conventional drying methods such as oven drying attesting to the greater FW content in the whole fruit confections180. It is also important to note, that even though BBPC may have a higher %FW when compared to BBEC, both confections remain below values that would lead to textural and microbial stability of these products as shown previously in a variety of food systems181. 55

3.3.3 Microstructural and macroscopic texture differences observed in blueberry extract and powder confections over one month storage

Microstructural differences in the gel matrices of the BBPC and BBEC were studied using rheological oscillatory strain sweeps. The inclusion of blueberry powder into the gelatin confectionary matrix produced a much firmer gel when compared to the blueberry extract inclusion confection (Figure 12), The storage modulus (G’) ranged from

3,000- 7,500 Pa in the BBPC to 800-2,000 Pa in the BBEC corresponding with previous findings for G’ values in gelatin gels182.

A 10000

1000 G'(PA)

100 0.01 0.1 1 10 100 1000 10000 OSCILLATION STRAIN (%) Day 0 G' Day 1 G' Week 1 G' Week 2 G' Week 3 G' Week 4 G'

B 10000

1000 G'(PA) 100

10 0.01 0.1 1 10 100 1000 10000 OSCILLATION STRAIN (%) Day 0 G' Day 1 G' Week 1 G' Week 2 G' Week 3 G' Week 4 G'

Figure 12. Rheograms of blueberry extract and powder confections over four week storage at 4°C. (A) Oscillatory strain sweep of BBEC at 25°C over four-week storage.

(B) Oscillatory strain sweep of BBPC at 25°C over four-week storage

The storage modulus remained higher in the BBPC over all six time points measured, showing that those confections exhibited a more solid-like gel structure than the BBEC. This finding could be due to the fact that gelatin hydrocolloid systems, are 57 reinforced by sugar co-solutes up to a peak concentration, after which the gel system begins to weaken due to lack of water available to be incorporated into the gel. In this application, BBPC’s gelatin network was reinforced by its moderate amount of added sugar (2%) , when in the case with BBEC containing 30% added sucrose, there was not adequate water to be incorporated into the gel matrix and the gel was weakened overall131,148. Pectin has also been shown to shorten the gelatin matrix resulting in a gel that has an intermediate property between the brittleness of pure pectin gels and elasticity of gelatin gels183. The BBPC gel displayed a gradual loss of linearity past the gel permanent deformation point (BBEC: 100% strain; BBPC: 10% strain) characteristic of a composite gel gelatin matrix 184.

While microstructural analyses on food gels are important for full characterization of molecular interactions, macroscopic deformations of the gels are relevant for how they relate to food texture properties, since large deformations occur in the mouth while chewing185,184. In this study, textural properties (hardness, cohesiveness, gumminess, and chewiness) of a blueberry powder and blueberry extract confection throughout one month of refrigerated are shown in Figure 13-16.

Figure 13. Hardness of BBEC and BBPC over four- week storage

Figure 14. Cohesiveness of BBEC and BBPC over four- week storage

Figure 15. Gumminess of BBEC and BBPC over four- week storage

Figure 16. Chewiness of BBEC and BBPC over four-week storage * Denotes a significant difference between inclusion type at the same time point (p≤0.05)

Lowercase letters denote a difference within a single inclusion across time (p≤0.05) 60

It could be seen in three of the four texture attributes measured (hardness, gumminess, and chewiness), that a one week reorganization, or curing period, was needed for the

BBPC to reach textural stability. Since the BBPC had a much lower sucrose content as discussed previously, the water within the gel network was more loosely bound than in the BBEC which allowed for the maintenance of a hydration layer around the polysaccharide/protein gel matrix which continued to reorganize for the first week of storage186. This “curing” period in which the powder confections underwent texture destabilization has been seen in other functional confections with whole powder inclusions in past work120,187, however usually it occurs much more quickly

(approximately 24 hours) and was reflected mainly in their moisture analyses. This work was the first to capture this complex reorganization of water through multiple parameters of texture profile analysis and throughout the first week of storage.

On Day 0 BBPC was significantly higher on Day 0 in Hardness (N): 178.8±28.0;

Gumminess (N): 124.7±12.8; and Chewiness (N): 1,097.2±112.9 when compared to

BBEC (Hardness: 85.9±19.0; Gumminess: 78.0±15.5; Chewiness: 686.2±136.7), but this trend was reversed in most parameters when readings were measured in day 1, and BBEC

(Hardness: 91.6± 18.5; Gumminess: 82.7±13.9; Chewiness: 727.7± 122.3) was significantly higher than BBPC (Hardness: 96.7± 30.1; Gumminess: 68.8± 16.0;

Chewiness:605.3 ±140.9). BBEC continued to be higher in all three parameters for the rest of the four-week storage. This finding was also supported by our DSC results in which the BBPC continued to have a reorganization of its internal water, although total moisture remained constant, throughout the entire month storage, while the BBEC remain

61 unchanged over the one month testing period. It is also important to mention that both the

BBEC and BBPC reached a hardness of 48.4 N and 59.5N at week 4, which has been shown to be near the ideal consumer acceptability range of 30-40 N in past confection texture studies120.

3.3.4 Sensory analysis of blueberry confections

The sensory attributes of two functional blueberry confections were evaluated by

75 participants (39% male, 61% female, 63% aged 18-25 years old, 37% above 25 years old); in order to compare the sensory acceptability of the two gelatin confections. No significant preference was observed between the blueberry powder (43/ 75 people preferred, 57%) and blueberry extract (32/ 75 people preferred, 43%) confections.

Panelists were split in their comments: those that looked for a true blueberry fruit flavor, and those that favored a “traditional, artificial” tasting blueberry flavor suggesting the lack of preference of one type of confection over the other. Both blueberry confections were over-all “slightly liked” by consumers in the acceptability tests (BBPC: 6.3±1.8,

BBEC: 6.2±1.7, Figure 17) as observed previously114,118,119,161.

Figure 17. Sensory acceptability of blueberry confections. A score of 1 corresponds to “Dislike Extremely” on the scale, while a nine corresponds with “Like Extremely”.

No significant difference in consumer acceptability was detected among aroma

(BBEC: 6.25±1.4; BBPC: 6.32±1.7), fruit flavor (BBEC: 6.48±1.5; BBPC: 6.99±1.5), sweetness (BBEC: 6.63±1.45; BBPC: 6.73±1.55), bitterness (BBEC: 6.03±1.43; BBPC:

5.79±1.83), or texture (BBEC: 5.85±2.32; BBPC: 5.83±1.99) when comparing the mean hedonic scores for the two different blueberry confections. Not surprisingly, the blueberry extract confection was found to be significantly more acceptable with regards to graininess (BBEC: 6.4±1.7; BBPC: 5.7± 1.2, p-value: 0.015) likely due to the particulate matter in the whole fruit confection. Overall both blueberry confections were

63 found to be sensory acceptable and are promising candidates for yielding high participant compliance in future clinical trials.

JAR testing revealed some opportunities for reformulation of each confection before moving forward for further testing, seen below in Figure 18 and Figure 19.

Fruit Flavor

Bitterness

Sourness

Sweetness

Firmness

Stickiness

Fruit Aroma

0 10 20 30 40 50 60 70 80

Much too little Slightly too little JAR Slightly too much Much too much

Figure 18. BBEC Just About Right analysis scores

BBEC JAR results suggest that the 75 panelists thought that the BBEC was too low with regards to fruit flavor and sourness, while on the contrary slightly too high in firmness and stickiness. The low ratings with regards to fruit flavor was unsurprising since the BBEC has been stripped of lots of natural blueberry volatiles through its extraction process. However, it was not expected for the BBEC to not receive JAR results for firmness and stickiness, since originally its texture seemed to mimic that of conventional gelatin confections better than BBPC. These attributes could be reformulated to increase consumer acceptance by adding some more blueberry flavor or

64 sugar to mask the present synthetic solvent notes, as well as decrease the a mount of gelatin in the formulation to try and decrease gummy stickiness and firmness.

Fruit Flavor

Bitterness

Sourness

Sweetness

Firmness

Stickiness

Fruit Aroma

0 10 20 30 40 50 60 70 80

Much too little Slightly too little JAR Slightly too much Much too much

Figure 19. BBPC Just About Right analysis scores

BBPC JAR testing revealed that consumers thought the whole powder confection was Just About Right for all tested attributes, but maybe also slightly too high with regards to sourness. This result is logical due to the fact that BBPC contained its inherent organic acids from the whole blueberries themselves, in addition to the added formula citric acid, while BBEC only contained the added acid. Overall it seems the BBPC may need less of a reformulation if chosen to move forward with a future clinical trial than

BBEC.

3.4 Conclusion

A direct comparison and characterization of a functional gelatin confection, one made with lyophilized blueberry powder and one made with an anthocyanin-rich 65 blueberry extract, over one month storage was achieved. Sensory analysis revealed no significant consumer preference between the two formulations, and thus full physico- chemical analysis of both confections through four weeks of refrigerated storage was necessary to add to the whole food vs. extract foods for health debate. Anthocyanin profile and quantification data revealed that while both the BBPC and BBEC contained the same 13 anthocyanin profile throughout storage, the BBPC retained more of its anthocyanin after confection manufacturing, and maintained a higher retention over the four weeks. However, when comparing the anthocyanin content of both confections from

Day 0 to week 4, no significant difference in anthocyanin degradation was found. Total moisture within both confections type remain stable throughout storage, however a greater amount of freezable water was found in the BBPC thought to be due to the higher water binding capacity of sucrose to pectin. With regards to the gel strength and microstructure, the synergistic gelling effect of the natural fruit pectin found in freeze- dried blueberry powder in addition to the gelatin added in the formulation as well as high sucrose levels in BBEC which bound the majority of water in the gel system, produced a stronger gel in BBPC in comparison to the BBEC. Finally, it was found in this experiment that a week-long water reorganization period was necessary for the BBPC to reach textural stability, while the BBEC texture parameters remained constant from Day

0- week 4 without much change. In conclusion, main differences between including a whole blueberry powder vs. an anthocyanin-rich blueberry extract with an equivalent gelatin confection matrix produced major differences with regards to bioactive retention, gel strength, texture, and freezable water content, but no significant differences in

66 consumer acceptability. Due to the stability of both functional confections over the four week storage, both would be excellent candidates for use in a future clinical trial for chemobrain mitigation, but future work is needed to compare bioactive uptake between a bioactive-rich extract and a whole fruit powder in an equivalent food matrix.

Chapter 4. Comparison of anthocyanin delivery after consumption of blueberry extract and whole blueberry powder confections in healthy men and women

4.1 Introduction

The consumption of a diet high in anthocyanins and other flavonoids has been associated with numerous health benefits such as obesity control, diabetes control152, cardiovascular disease (CVD) prevention153, cancer prevention,154 and improvement of visual and brain function.155 Though numerous fruits have been shown to have high anthocyanin content, such as raspberries, blackberries, and red currants 188, blueberries were studied due to their high consumer acceptance, regular availability, and ample literature evidence with regards to their health benefits; particularly in cognitive health.

Treatment with blueberries or blueberry extracts have demonstrated potent cognitive improvement in in vitro cell culture studies157, in vivo rodent models103,104,106,158, and in human studies107,108,159 across a plethora of cognitive disorders and thus these preliminary findings substantiate the need for a fully characterized and highly acceptable delivery vehicle of blueberry bioactives to be formulated and tested for use in future cognitive health clinical trials. Current gaps in the blueberry and cognitive health literature include accurate quantification of blueberry bioactive dose, measurement of participant compliance to the blueberry treatments, measurement of individual bioactive uptake after consumption, as well as a direct comparison of blueberry bioactive delivery and uptake between whole lyophilized blueberry powder and anthocyanin-rich blueberry extract when delivery matrix and total anthocyanin dose are controlled for.

Cognitive disease can range anywhere from Mild Cognitive impairment (MCI), in which patients experience subtle memory complaints to Alzheimer’s Disease (AD) which in 2016 was the sixth leading cause of death in the United States189,190. It was estimated that in 2006, around 26.6 million people in the world were living with AD, with the hypothesis that that number will quadruple by the year 2050 to affect 1 in every 85 people191. A lesser known cognitive disease whose instance is also exponentially increasing is the cognitive decline experienced as a side effect of receiving chemotherapy known as chemotherapy induced cognitive impairment (CICI). CICI, colloquially known in the medical community as “chemobrain” can be defined as a state of prolonged cognitive dysfunction resulting from administration of a variety of common cytotoxic chemotherapeutic agents109. The symptoms of CICI include memory loss and depression and can be some of the most debilitating cancer survivorship issues that patients face after treatment109. Chemobrain is thought to affect up to 78% of patients who undergo chemotherapy, with no current treatment available. While it seems that the incidence of cognitive disorders is continuing to rise globally, it is hypothesized that these maladies, such as CICI, can be delayed and/or mitigated with early detection and intervention189.

This hypothesis makes the CICI a promising candidate cohort for the study of anthocyanin-rich nutritional interventions and their potential to serve as preventative or adjuvant therapies for those receiving chemotherapy or suffering from cognitive decline as a result of CICI.

A 2010 study performed by the National Cancer Institute estimates that around

75% of adults in the U.S. do not consume enough fruits and vegetables to meet the

69 minimum recommended amounts by MyPyramid, and that over 90% of Americans had intakes of empty calories that exceeded the discretionary calorie allowances192. This coincides with the fact that Americans consume up to 24% of their daily energy from snack foods193, thus presenting an opportunity for the development of anthocyanin rich functional snacks for the successful delivery of fruit phytochemicals as well as provide a medium to compare the bioactive delivery capacities of whole fruits vs. their extracts in a controlled manner . The objective of this study was to compare flavonoid delivery in 24 hour urine, safety, and sensory acceptability of 2 blueberry- based amorphous functional confections (one containing blueberry extract and the other of a whole blueberry powder) formulated to deliver the 125 mg of anthocyanins per serving for use in future CICI clinical studies in a cross-over intervention study performed on healthy adult men and women. We hypothesize that there will be no difference in anthocyanin uptake or delivery between the BBEC and BBPC in 24 hour urine samples due to equivalent dose administered and matrix used.

4.2 Materials and Methods

4.2.1 Blueberry confection preparation

Whole blueberry powder (N1112 Blueberry Powder) and blueberry extract

(N1077 VitaBlue) used as inclusions in the gelatin confections used in this study were made from the same raw materials (Vaccinium corymbosum) and purchased from the same supplier (Futureceuticals, Momence, IL). Both confections were prepared by mixing gelatin (Knox Gelatin, Treehouse Foods, Inc., Oakbrook, IL), sugar (Domino

Foods Inc., Iselin, NJ), citric acid (Tate and Lyle, Decatur, IL), Jell-O Berry Blue flavor

(Kraft Foods, Northfield, IL), and either the blueberry extract or powder described previously. This mixture was then stirred and heated on a hot-plate until reaching 100C, which typically took approximately 20 minutes. Upon reaching boiling, the confection base was removed from heat and moved to a 9 X 9 inch square baking pan lined with parchment paper subsequently placed in refrigerator (4 C) to solidify, which took approximately 3 hours. Confections were then removed from the pan and cut into squares

(1in x1in x0.5 in), placed in Ziploc bags, and stored at 4 C, avoiding air and light, to mimic how the confections would be stored if used in a clinical trial. The final formulations of blueberry extract (BBEC) and blueberry powder (BBPC) confections are presented in Table 1. Both confections were initially formulated to deliver approximately

320 mg of anthocyanins/ 100 g dose ( in past work, the extract (115.29±0.43 mg anthocyanins/ g extract) was found to be approximately ten times as concentrated as the whole food powder (10.83±0.53 mg anthocyanins/ g powder)) with regards to anthocyanin content, or about ten confections, which is equivalent to eating 2 cups of fresh blueberries12. However, it was also found that even though anthocyanin content was originally formulated to be equivalent, the bioactives present were retained differently after confection manufacturing between the BBEC (2.09 mg anthocyanins/ g confection) and the BBPC (2.83 mg anthocyanins/ g confection). Therefore to maintain a constant dose of anthocyanins in this preliminary clinical intervention, the number of confections consumed varied between the BBEC (6 confections, ~60 g) and BBPC (4 confections,

~44 g).

Table 3. Blueberry extract and powder confection formulations

Blueberry Extract Blueberry Powder Ingredient Confection (%) Confection (%) Water 37.0 37.0 Sugar 29.3 2.0 Jello Berry Blue 26.0 26.0 Knox gelatin 4.5 4.5 Citric acid 0.5 0.5 Blueberry Extract 2.8 0.0 Blueberry Powder 0.0 30.0 total 100.0 100.0

4.2.2 Anthocyanin delivery clinical study

This study protocol was approved by the Institutional Review Board at The Ohio

State University (2018C0041), and participants provided written informed consent before enrolling in the study. 12 Healthy men (n=6) and women (n=6) were enrolled in this pilot study based on age (18- 65 years), body mass index (18-35 kg m-2), non-smoking status

(adults who have never smoked or who have not had a cigarette in the past ten years), non-use of antibiotics (>6 months), alcohol consumption (< 2 drinks per day), freedom from digestive, metabolic, or immunologic disorders, as well as free from food intolerances to study products (blueberries), other berries, and gelatin, as well as not being a vegan. In a randomized, crossover design, participants arrived to the study center after 7 days of following a low berry diet (Appendix A) to ingest one serving of either the blueberry extract (approximately 60 g confection / dose) or blueberry powder confection

(approximately 44 g confection/ dose) containing approximately 125 mg anthocyanins/

72 dose (Table 3) on two occasions separated by a second 7 days of the low berry diet after which they would consume the other type of confection. 6 of the 12 participants were randomized to receive the blueberry extract confection in the first arm of the study

(Figure 20). 24 hour urine was collected throughout the day immediately after consumption of each of the blueberry confections and returned to the study coordinators the following day, for a total study duration of 16 days.

Figure 20. Clinical design

On the first visit (day -7), participants began the informed consent process and all study procedures were thoroughly explained. Additionally, participants were instructed by the Study Coordinator on how to follow a controlled low berry diet and were educated on completing health history forms, daily compliance forms, and diet questionnaires for the study (see Appendix). After the washout, subjects returned to the study center for the 73 day 0 randomization to one of the two different blueberry arms. The subjects provided a spot urine upon arrival to confirm compliance to the low berry diet and monitor baseline anthocyanin levels. The study coordinator then distributed the berry confections and sensory questionnaire (see Appendix A). Each participant received their assigned blueberry confections containing 125 mg anthocyanins (approximately 6 extract confections and four powder confections due to the previously mentioned differences in anthocyanin retention after confection manufacturing). Participants consumed all confections during the visit, which was equivalent to ~ 1 cup or 2 servings of fresh blueberries11. Once the confections had been completely consumed, the 24-hour urine collection began. Participants left the study center with a 24-hour urine collection container and instructions for urine collection. The following day (Day 1) the participant returned the urine container to the study center and then resumed the low-berry diet for another week. Participants were asked to complete a three-day diet record for two days prior to each intervention and the day of the intervention (days -2, -1 and day 0 as well as day 6, 7 and 8). The diet records were turned in to study staff at the same time as the 24- hour urine collection. Study procedures were then repeated on Day 8 with the crossover.

4.2.3 Sensory analysis

Participants completed a sensory analysis of their assigned blueberry confection during the first visit of their intervention. Sensory tests were administered to participants in a private room while seated in chair under fluorescent lighting. Participants received the entire serving of either the BBEC (60g, or 6 10 g confections) or BBPC (44 g, or 4 11

74 g confections) and were asked to complete their sensory analysis as they ate the entire serving.

Paper surveys designed with a 9-point hedonic scale (1 =dislike extremely to 9 = like extremely) were administered to assess acceptability of the confection, overall liking, fruit aroma, fruit flavor, sweetness, texture (hardness), grittiness (sandiness), and bitterness. A 5-point just- about-right scale (JAR; 1 = much too weak to 5 = much too strong) assessed flavor (fruit, sweet, sour, and bitter), aroma (fruit), and texture (firmness and stickiness) in order to guide future optimization of the functional confections.

Participants were also asked general impression questions about the confections which included attitudes about how many and how often they would be willing to consume fruit confections.

4.2.4 Dietary control and analysis

Participants were instructed by the study team to abstain from a list of berry and berry products for seven days prior to the consumption of both of the blueberry confections to isolate anthocyanins delivered to the participant solely from the confection and avoid any interference that may be experienced with the anthocyanin urinary analysis. Participants were also asked to abstain from other polyphenol rich foods such as red and white wines, black-eyed peas, black beans, red or purple fruits and vegetables, and certain nuts (see Appendix for full list). In addition to the seven day low- berry diet, clinical participants were also instructed to complete a 3 day dietary recall for two days prior and the day of their intervention, which was verified by a registered dietician or study personnel for accuracy. The same dietary restriction measures were employed for

75 the second phase of the crossover study. Energy and nutrient intakes were assessed using the Nutrition Data System for Research dietary analysis software (University of

Minnesota; Version 2014; Minneapolis, MN).

4.2.5 Anthocyanin urinary analysis

24-hour and spot urine samples were aliquoted into 2mL cryovials after collection at the CRC. These aliquots were then frozen and stored at -80°C until UHPLC-MS/MS analysis could take place at the Nutrient and Phytochemical Analytic Shared Resource

(NPASR) here at The Ohio State University. At the time of analysis urines were thawed and acidified with formic acid to 5%v/v and filtered through 0.2µm PTFE syringe filters.

Acidified and filtered urine was then injected (20uL) on a UHPLC-MS/MS system

(Agilent 1290 Infinity II UHPLC, 6495 QqQ triple quadrupole) and interfaced with positive ion electrospray. Mobile phase (A) consisted of 5%v/v formic acid in water and

(B) was 5%v/v formic acid in ACN. After injection the mobile phase composition was held at 100%A for 1.5min followed by a linear gradient to 18.5%B by 15min, increased to 95%B by 16min and held for 1min before returning to the initial condition over 3min.

Total run time was 20min at a flow rate of 0.3mL/min and 40 °C column temperature.

The MRM method was developed using blueberry anthocyanins from blueberry powder providing both expected MS/MS transitions and diode array UV-vis spectra. This provided confirmation of the identities of the vast majority of anthocyanins measured in urine. Additional transitions were designed according to common fragmentation of glucuronide conjugates, i.e. a 176 amu neutral loss from the precursor ions. In this way transitions were generated for delphinidin, cyanidin, petunidin, peonidin, and malvidin

76 glucuronides. A collision energy of 25 eV was determined optimal for both the precursor anthocyanins from blueberry as well as the tentative glucuronide metabolites.

Corresponding UV-vis peaks at 520nm were observed to corroborate the glucuronide identities in alignment with the respective MS/MS transitions.

Calibration was performed with cyanidin 3-O-glucoside standard (Indofine

Chemical Co., NJ). The standard was dissolved in water containing 0.1M HCl and absorbance of stock recorded at 511nm. An extinction coefficient of 25,740 was used

(McClure, 1967) to determine the stock concentration. Dilutions were made from this stock into 5%v/v formic acid in water to provide standard curve solutions bracketing the range of metabolites detected.

4.2.6 Statistical Methods

Data were analyzed by SPSS (IBM, New Castle, NY). All results were considered statistically significant at a p-value ≤0.05. JAR results were displayed qualitatively as stacked bar charts. Paired t-tests were used to compare mean acceptability scores of the confections by the clinical participants. Total anthocyanin concentrations in 24-hour urine after consumption of one blueberry treatment will be compared to each participant’s baseline urine and to total anthocyanins after consumption of the other blueberry treatment in a paired t-test (p≤0.05).

4.3 Results and discussion

4.3.1 Participants

A total of 12 healthy participants (6 male, 6 female) were enrolled and completed the study without any adverse events during the sensory evaluation or the crossover

77 clinical study. Nearly all participants were of normal weight according to BMI (18.5-25), with only one of the twelve participates falling into the overweight range (<30), with the mean BMI being 25.0±2.8 kg/m2 194 (Table 4. Demographic and compliance data of clinical cohort). The average age of the clinical cohort was 27 ±8 years, and the majority followed healthy lifestyles such as consuming at least one serving of anthocyanin-rich fruits and vegetables per week (91.7%) as well as exercising for at least thirty minutes once/ week (75.0%). High compliance was also observed during the 7 day low- anthocyanin washout period prior to both intervention periods (BBEC washout compliance: 85.7±12.2% BBPC washout compliance: 86.9±11.3%).

Table 4. Demographic and compliance data of clinical cohort

Age (mean ± SD) 27 ±8 years

BMI (mean ± SD) 25.0±2.8 kg/m2

Regular anthocyanin food consumers (≥1 91.7% (11/12)

to 3 servings/ week)

Regular exercisers (≥30 min/ week) 75.0% (9/12)

Washout Diet Compliance (mean ± SD)

Blueberry extract intervention 85.7±12.2%

Blueberry powder intervention 86.9±11.3%

NDSR analysis of the 3-day dietary records obtained throughout both blueberry confection interventions showed no significant difference in the overall amount of food consumed, total calories, fat, carbohydrates, protein, or fiber among the 12 participants between their first and second blueberry confection intervention. Throughout the 16 day trial, participants consumed an average of 1753.9±308.0 g of food/ day containing

2,123.8± 571.9 calories, 104.7±39.8 g fat (45% of calories), 204.8±60.2 g carbohydrates

(39% of calories), 86.7± 29.5 g protein (16% of calories), and 21.8±7.0 g total dietary fiber, which were similar to the dietary reference intakes (DRIs) for the age, gender, and activity breakdown of the clinical cohort except for protein intake, which was higher in this work than was recommended by the U.S. Department of Agriculture195 . Average breakdown of these dietary parameters for each participant and intervention can be found in Appendix A.

4.3.2 Sensory analysis of blueberry confections

Due to the small sample size of this clinical sensory data, its analysis was used as a qualitative comparison agent for the larger consumer sensory performed in the previous chapter, rather than designed as a statistically rigorous standalone experiment. In this data, as was seen in the consumer sensory, both the BBEC and the BBPC were liked by the clinical participants and scored above neutral in the hedonic testing (Figure 21).

10 9 8 7 6 5 4 3 2 1 0 Overall Aroma Fruit Flavor Sweetness Texture Grittiness Bitterness Liking (hardness) (sandiness)

BBEC BPPC

Figure 21. Sensory acceptability of blueberry confections used in clinical trial A score of one corresponds to “Dislike Extremely” on the scale, while a nine

corresponds with “Like Extremely”.

Results indicate that there was no significant difference among any of the attributes that were asked about in the sensory test between BBEC (Overall liking: 7.1±0.9, aroma:

5.8±1.6, fruit flavor: 6.9±1.2, sweetness: 6.6±2.0, texture (hardness): 6.3±1.7, grittiness

(sandiness):7.0 ±1.4, and bitterness: 6.25 ±1.5) and BBPC (Overall liking: 6.5±2.0, aroma:6.2 ±1.9, fruit flavor: 6.9±1.9, sweetness: 6.7±2.0, texture (hardness):6.75±1.6, grittiness (sandiness): 5.1±2.4, bitterness:6.0±2.2). Overall, the scores obtained in this sensory test coincided with those obtained in the consumer sensory test performed during the development of the confections. This similarity in sensory scoring suggests than consumers enjoyed the taste of the confections equally as much when they were aware and when they were not aware of a possible health benefit that may be associated with consuming the confections, which is surprising based on previous literature with functional food products196. This could be due to the marketing principle that even though 80 nutritional information can influence product liking and intent to buy by consumers, the experience with the product itself is the main driver of consumer hedonic scores197. It is also likely that a traditionally consumer acceptable and quickly growing delivery vehicle, such as a gummy confection, would drive positive sensory scores regardless of whether or not a health benefit is associated198.

JAR scores suggest that the BBEC were slightly too firm according to consumers, as well as slightly too low in both fruit flavor and fruit aroma, which also coincided with our group’s previous sensory results (Figure 22). Firmness could be adjusted in future work by decreasing the amount of gelatin added to the formulation, however the fruit aroma and taste may be difficult to increase, since the majority of characteristic and fruit- like blueberry flavor compounds are nonpolar and most likely lost in the polar extraction of the berries at the producer before purchase199.

Firmness

Stickiness

Fruit Aroma

Bitter

Sour

Sweet

Fruit Flavor

0 2 4 6 8 10 12

Much too little Slightly too little JAR Slightly too much Much too much

Figure 22. BBEC Just About Right analysis scores

JAR scores for BBPC suggest high levels of overall satisfaction with the confection by clinical participants, even though it’s overall liking score was found to be slightly lower than the BBEC (Figure 23). This suggests that not much reformulation would need to be done if utilizing this confection in a larger clinical study looking at a cognitively impaired cohort.

Firmness

Stickiness

Fruit Aroma

Bitter

Sour

Sweet

Fruit Flavor

0 2 4 6 8 10 12

Much too little Slightly too little JAR Slightly too much Much too much

Figure 23. BBPC Just About Right analysis scores

In addition to promising sensory scores being obtained for the two blueberry confections, participants were also asked general questions about how frequently they could see themselves consuming such a confection if asked to participate in another clinical study utilizing them. Participants said they could consume both of these confections anywhere from one to four times/ day for up to 2 months (Table 5). These results reaffirm the likelihood of high compliance for both the BBEC and the BBPC if used in a future larger clinical trial as reported in previous clinical studies using functional confections114,118,119. This data will also provide guidance for choosing the 82 amount of confections and the length of a future clinical trial to maximize both dose efficacy and ecological validity, which are important factors that are often overlooked in nutritional clinical trial design200.

Table 5. Consumption frequency of blueberry confections

Question I could consume two I could consume four

confections … confections every day

for…

Responses BBEC BBPC Responses BBEC BBPC

Not at all 0 1 Not at all 0 1

4 times/ day 4 6 3 days or less 2 0

2 times/ day 3 3 1 week or more 3 5

1 time/ day 4 2 1 month 2 1

1 time/ week 1 0 2 months or 5 5

4.3.3 Anthocyanin levels in urine

When analyzing the spot urine samples that were taken after the one- week low berry diet washout period and before each confection intervention, no peaks appeared when monitoring at 520 nm, confirming their compliance with the diet (Figure 24). These results coincided with the self- reported compliance from the participants themselves, who on average reported that they did not consume any of the prohibited anthocyanin- containing foods for approximately 6 out of the 7 assigned washout days (BBEC:

85.7±12.2% compliant BBPC: 86.9±11.3% compliant) (Table 4). It was not surprising that a one- week anthocyanin consumption abstinence resulted in no urinary anthocyanins being detected, since anthocyanins have been shown to predominantly leave human urine approximately 4 hours after consumption201.

24 hour urine samples from all 12 participants after both BBEC and BBPC intervention showed that all of the 13 anthocyanins present in the confections were also found in their parent forms. Glucuronides of all of the 5 parent anthocyanidins

(delphinidin, cyanidin, petunidin, peonidin, and malvidin) were also detected in human urine, however only cyanidin glucuronides have been seen reliably in previous work as metabolic derivatives of anthocyanins after human consumption201. In concentration, total anthocyanins and anthocyanin conjugates detected in the 24 hour urine aliquots were averaged across all 12 participants resulting in 100.14±33.3 nmoles for BBEC and

107.3±90.8 nmoles for BBPC. This is similar with concentrations seen in past anthocyanin interventions, in which parent anthocyanins are not very stable in 24 hour urine, and can only be quantified at approximately 0.05% of the ingested dose, with the majority of anthocyanin consumption markers being categorized as anthocyanin degradant products201.

When comparing the anthocyanin delivery of the means of all 12 participants after consuming 125 mg of anthocyanins from either the BBEC or BBPC, there was no significant difference among the two treatments, which is also not surprising due to the small sample size and great individual variability of people’s metabolism of plant phytochemicals 202. This variability is confirmed by the fact that one participant excreted

147 nmoles of anthocyanins from BBEC and 361 nmoles anthocyanins from BBPC after the acute ingestion while another only excreted 58.6 nmoles from the BBEC and 114.67 from BBPC of the formulated 125 mg anthocyanins. However, when comparing the uptake of anthocyanins from BBEC vs. the BBPC within one subject, values remained relatively consistent, except in participant 11, who absorbed almost 2.5 times more anthocyanins from the BBPC than the BBEC treatment arm (Figure 24). The aliquot of

Participant 14’s 24 hour urines were lost to filtering and will be reanalyzed in the future.

400

350

300

250

200

150 (nmoles) 100

0 0 1 2 3 4 5 10 11 12 13 15 24 hour 24 urineAnthocyanin concentration Participant

BBEC BBPC

Figure 24. Urinary recovery of BBEC and BBPC anthocyanins in 24 hour urine samples

Since it is now known that the BBEC and BPPC can deliver approximately 0.05% amount of anthocyanins that they are formulated for in this matrix, time frame and dose level, more work needs to be done to determine what is the circulating concentration needed to exert effectiveness in the symptoms of cognitive decline experienced by CICI sufferers. It would also be interesting to examine how much of an increase in formulated

85 anthocyanin levels would correlate with an increase in in urinary anthocyanin excretions as well as if this relationship is linear and/ or can be saturated.

4.4 Conclusion

In conclusion, after a cross over study in which 12 healthy men and women consumed two different but equivalent doses of blueberry anthocyanins (one coming from a lyophilized whole fruit powder and one coming from an extract) within a gelatin confection matrix, no difference in bioavailability was seen in 24 hour urine samples.

Analysis of spot urine samples confirmed their compliance to the week-long low berry washout diet, and analysis of their total food intake for the two days prior and day of intervention suggest that these findings may stem from the anthocyanin source and no other external factors. Sensory analysis of both confections provided by the clinical participants showed no significant difference in hedonic liking of several key confection attributes. Overall, both the BBEC and BBPC would be successful functional food delivery vehicles for blueberry anthocyanins in future clinical trials looking at the cognitive decline associated with CICI. This work also holds food science and nutritional significance in functional food product development by demonstrating preliminary evidence of equal bioavailability of a whole fruit powder and a phytochemical-rich extract in an equivalent food matrix.

Chapter 5. Conclusions and future work

Results from this study demonstrate that both physico-chemical properties, behavior throughout one month of storage, and bioactive delivery is affected by the inclusion of lyophilized blueberry powder vs. anthocyanin-rich blueberry extract in a gelatin confection matrix. Although there was no significant consumer preference between the BBEC and BBPC, differences were seen with regards to bioactive retention after confection manufacturing, with BBPC retaining a higher level of anthocyanins per gram of confection at Day 0 and over 4 weeks refrigerated storage. BBPC also had a much greater level of freezable water throughout storage attributed to the higher water binding capacity of sucrose, the main water binder in the BBEC, when compared to fruit pectin, found in the BBPC. The BBPC produced a stronger and more stable gel microstructure due to the lack of water availability in the BBEC, and these differences were also reflected in the texture analysis of the confections over time. BBPC was significantly harder than BBEC at Days 0 and 1, however, interestingly decreased in hardness after one week storage, where BBEC became the harder confection. Overall, although differences were observed in bioactive resistance to degradation throughout the confection manufacturing process, amount of freezable water, strength of gel microstructure, and gel texture attributes when comparing BBEC and BBPC, ultimately both confections were deemed to be successful in a future clinical trial due to the stability in their individual shelf-life parameters and lack of significant sensory preference among consumers.

BBEC and BBPC were examined to assess differences in bioactive delivery to healthy clinical participants. During a cross-over study of 12 healthy men and women in which all participants ate a single equivalent acute dose of both the BBPC and BBEC controlled for anthocyanin content 24 hour urine and spot urine samples were collected.

Results indicated that participants were compliant with their 7 day low berry washout diets and no significant difference in absorption of the blueberry anthocyanins was detected when comparing treatments. Review of three-day dietary records that were recorded for two days prior and the day of the two confection interventions showed participant background diet values that were consistent for calories, fat (g) , carbohydrates (g), protein (g), and fiber (g) with the DRIs for the age, gender, and activity level of the clinical cohort being studied in this intervention. It was also seen that there were no significant differences for any of the selected dietary parameters between the two intervention periods, suggesting a lack of contribution of diet composition to bioactive uptake. Sensory questionnaires were also given to clinical participants, to compare if there was any differences in product liking when comparing a clinical consumers vs. non-clinical consumers. Results indicated that, similar to the developmental consumer sensory, there was no significant differences in liking scores between the BBEC and BBPC in the clinical context, however BBPC performed better in the JAR analysis than the BBEC and may not need reformulation if chosen to move onto a larger clinical trial in a cognitive dysfunctional cohort. Responses about how frequently clinical trial participants would be willing to consume fruit based confections confirmed

88 the feasibility of using the BBEC or BBPC in a two month clinical trial and maintaining high participant compliance.

For future work, a NMR metabolomics study will be performed on the existing clinical 24-hour urine samples to determine if there are any changes in a person’s global urinary metabolome when eating full lyophilized blueberry powder or a blueberry extract.

Also, a larger clinical trial will be necessary to determine dose/ response information of the confections which would also require some scale up studies to ensure that the blueberry confections remain stable when produced in large quantities and not at the benchtop scale, which was done in this work. It would also be interesting to lower the calorie content of the confections by adding alternative sweetening agents to the formulation in replacement if sucrose (i.e. high intensity sweeteners) and look at its effect on the shelf stability and physico-chemical properties of the confections, as well as determine if there would be any negative interference of these additives with the bioactivity of the blueberry inclusions or interaction with the gut microbiome. Other future work would be utilizing these blueberry confections in a large randomized control study in individuals suffering from chemobrain before and after chemotherapy treatment, and seeing if they impart any significant cognitive effect vs. a control intervention.

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Appendix A: Blueberry gummy clinical documents

A1. Study informed consent form

The Ohio State University Consent to Participate in Research

Study Title: Comparison of bioavailability and urinary metabolite profile after consumption of blueberry extract and whole blueberry powder confections in healthy men and women

Researcher: Yael Vodovotz, Ph.D.

Sponsor: None

 This is a consent form for research participation. It contains important information about this study and what to expect if you decide to participate. Please consider the information carefully. Feel free to discuss the study with your friends and family and to ask questions before making your decision whether or not to participate.  Your participation is voluntary. You may refuse to participate in this study. If you decide to take part in the study, you may leave the study at any time. No matter what decision you make, there will be no penalty to you and you will not lose any of your usual benefits. Your decision will not affect your future relationship with The Ohio State University. If you are a student or employee at Ohio State, your decision will not affect your grades or employment status.  You may or may not benefit as a result of participating in this study. Also, as explained below, your participation may result in unintended or harmful effects for you that may be minor or may be serious depending on the nature of the research.  You will be provided with any new information that develops during the study that may affect your decision whether or not to continue to participate. If you decide to participate, you will be asked to sign this form and will receive a copy of the form. You are being asked to consider participating in this study for the reasons explained below.

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1. Why is this study being done?

You are being asked to participate in this research study because blueberries contain several compounds which may be beneficial for human health and prevention of disease. These compounds can be consumed as part of a complex matrix in the whole fruit or also in a much simplified matrix in the form of an extract of the fruit. However, this relationship between bioactive source and uptake by humans after consumption is poorly understood and somewhat controversial. This study will look at a group of compounds found in blueberries called “anthocyanins” and how well they are absorbed into your body from 2 different types of blueberry confections made here at The Ohio State University. By studying the absorption and metabolism of these compounds, we may be able to understand how they influence health and disease, as well as which of the 2 confections is most easily absorbed.

2. How many people will take part in this study?

A total of 12 healthy men and women, all between the ages of 18 and 65, will be recruited for participation in this study.

3. What will happen if I take part in this study?

If you decide to participate in this study, the study coordinator will explain all details of the study and answer any questions you might have. You will then be randomly assigned (like the “flip of a coin”) to one of two different confection groups for a total of 16 days (one week of low-berry washout and one day of the berry confection and then repeated with a second one-week washout and one day of the second berry confection). The study will require that you make five visits to The Ohio State University Clinical Research Center (CRC), part of the Center for Clinical and Translational Sciences or the Parker Food Science Building.

Briefly, your study visits will be as follows. Additional details about each study visit can be found in the text below.

Day – 7 Parker Food Science Building  Begin informed consent process  Instruction on all study procedures and washout diet Day 0 CRC Visit  Baseline urine collection  Consume confections  Sensory questionnaire  Receive instruction on 3 day diet record  Receive 24-hour urine collection containers Day 1 Parker Food Science Building 104

 Return 24 hour urine collection to study coordinator Day 8 CRC Visit  Baseline urine collection  Consume confections  Sensory questionnaire  Receive instruction on 3 day diet record  Receive 24-hour urine collection containers Day 9 Parker Food Science Building  Return 24 hour urine collection to study coordinator

At the first visit (day -7), we will instruct you on how to follow a berry- free diet for the next 16 days, and how to complete the daily compliance forms and questionnaires. After the 1 week washout period, you will return to the CRC for the day 0 randomization to 1 of the 2 different blueberry confection groups. At that time, the CRC staff nurses will take your baseline urine and provide you with a 24-hour urine container and instruct you how to use it. The study coordinator will give you one serving of the blueberry confection group you were placed in first. Additionally, during this visit we will ask you to complete a one-time sensory questionnaire which you will complete after eating one serving of the confections. The purpose of the questionnaire is to give your opinion about the various types of confections. The sensory analysis will take approximately 15 minutes to complete. You will then be able to leave the CRC and complete your 24-hour urine collection to be returned to the Parker Food Science Building the next day. At the next visit to the CRC on Day 8, the CRC staff nurses will again take your baseline urine sample upon your arrival, and the study coordinator will give you one serving of the other blueberry confection along with the sensory questionnaire, and your second 24-hour urine collection container. You will consume the confections, complete the sensory evaluation, and return the 24-hour urine collection the following day.

4. How long will I be in the study?

You will be in the study for a total of 16 days.

5. Can I stop being in the study?

You may leave the study at any time. If you decide to stop participating in the study, there will be no penalty to you, and you will not lose any benefits to which you are otherwise entitled. Your decision will not affect your future relationship with The Ohio State University.

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If, at any time, you do withdraw from the study, the following FDA guidelines are in place:  According to FDA regulations, data collected about the participant up to the time of withdrawal remains part of the study data and may not be removed  Participants may be asked if they wish to provide informed consent for continued follow-up and data collection subsequent to their withdrawal from the interventional portion of the study  Data collected about the participant prior to withdrawal may be reviewed by the researcher and research team (including review of public records, such as survival status) even if the participant does not provide consent to continued follow-up.

6. What risks, side effects or discomforts can I expect from being in the study?

The physical risks associated with this study are believed to be low. There are no known side effects of any of the diets in this study. The berry confection consumed may have some gastrointestinal side effects such as diarrhea, but no other side effects are expected. The dose used in this study has been demonstrated to be safe in previous human research.

 Though it is a natural product, the effect of these berry confections on an embryo or fetus is currently unknown. You cannot take part in this study if you are pregnant or breast-feeding a child. You must agree not to become pregnant while you are in this study.

7. What benefits can I expect from being in the study?

You likely will not benefit directly from participating in the study, as the benefits of the blueberry products are unknown and modest at best over such a short period of time. Rather, the greatest benefits likely will be to the world of scientific research and the greater public with regards to information about berry products’ effect on health and disease.

8. What other choices do I have if I do not take part in the study?

You may choose not to participate without penalty or loss of benefits to which you are otherwise entitled. You might be eligible to participate in other clinical trials, and you can discuss these options with your doctor.

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9. Will my study-related information be kept confidential?

Efforts will be made to keep your study-related information confidential. However, there may be circumstances where this information must be released. For example, personal information regarding your participation in this study may be disclosed if required by state law.

Also, your records may be reviewed by the following groups (as applicable to the research):  Office for Human Research Protections or other federal, state, or international regulatory agencies;  U.S. Food and Drug Administration;  The Ohio State University Institutional Review Board or Office of Responsible Research Practices;  The sponsor supporting the study, their agents or study monitors

Participant information from studies involving drugs, biologics, or devices will be entered into the publicly available databank at ClinicalTrials.gov. However, all personal data will be de-identified prior to being entered into this website.

10. What are the costs of taking part in this study?

There are no costs related to participation in this study.

11. Will I be paid for taking part in this study?

$25 will be given to you after each of the four visits to the CRC where you will ingest a single serving of the confections, complete a sensory questionnaire, and collect and return 24-hour urine (Days 0&1 and Days 8&9). This is a total of $100. If you withdraw from the study at any time, you will receive money to compensate you for the time you were enrolled. Additionally, you will be given a parking pass for each visit.

12. What happens if I am injured because I took part in this study?

If you suffer an injury from participating in this study, you should notify the researcher or study doctor immediately (Steven K. Clinton, MD,

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PhD) at 614-293-2886, who will determine if you should obtain medical treatment at The Ohio State University Medical Center.

The cost for this treatment will be billed to you or your medical insurance. The Ohio State University has no funds set aside for the payment of health care expenses for this study.

13. What are my rights if I take part in this study?

You may refuse to participate in this study without penalty or loss of benefits to which you are otherwise entitled. If you are a student or employee at Ohio State, your decision will not affect your grades or employment status.

If you choose to participate in the study, you may discontinue participation at any time without penalty or loss of benefits. By signing this form, you do not give up any personal legal rights you may have as a participant in this study.

An Institutional Review Board responsible for human subjects research at The Ohio State University reviewed this research project and found it to be acceptable, according to applicable state and federal regulations and University policies designed to protect the rights and welfare of participants in research.

14. Who can answer my questions about the study?

For questions, concerns, or complaints about the study, or if you feel you have been harmed as a result of study participation, you may contact the Principal Investigator, Dr. Yael Vodovotz, at (614) 247-7696. You may also contact one of the study coordinators: Dr. Beth Grainger can be reached at (614) 293-7817, and Meredith Myers can be reached at (513) 569-2188.

For questions about your rights as a participant in this study or to discuss other study-related concerns or complaints with someone who is not part of the research team, you may contact Ms. Sandra Meadows in the Office of Responsible Research Practices at 1-800-678-6251.

Signing the consent form I have read (or someone has read to me) this form and I am aware that I am being asked to participate in a research study. I have had the opportunity to ask questions and have had them answered to my satisfaction. I voluntarily agree to participate in this study. 108

I am not giving up any legal rights by signing this form. I will be given a copy of this form.

Printed name of subject Signature of subject

AM/PM Date and time

Printed name of person authorized to consent for subject Signature of person authorized to consent for subject (when applicable) (when applicable)

AM/PM Relationship to the subject Date and time

Investigator/Research Staff

I have explained the research to the participant or his/her representative before requesting the signature(s) above. There are no blanks in this document. A copy of this form has been given to the participant or his/her representative.

Printed name of person obtaining consent Signature of person obtaining consent

AM/PM Date and time

Witness(es) - May be left blank if not required by the IRB

Printed name of witness Signature of witness

AM/PM Date and time

Printed name of witness Signature of witness

AM/PM Date and time

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A2. Blueberry gummy clinical informational flyer Blueberry Confection Study

Researchers at The Ohio State University Comprehensive Cancer Center are investigating the absorption of plant compounds in two types of blueberry confections.

Who can join this study? If you’d like to participate, you must:  be over the age of 18  have a BMI between 18 and 35  be a non-smoker  have no allergies to berries, wheat, or soy  consume no more than two alcoholic drinks a day

What will I be asked to do?  participate in a screening phone call for the study  agree to limit berry and berry foods in your diet for 16 days  eat two investigational berry confections over the 16 days  visit the OSU Clinical Research Center 5 times over the 16 day period for confections and urine collection

How do I learn more about the study? If you’d like to hear more about the study or are interested in participating, please contact one of our study coordinators: Meredith Myers, BS (513)-569-2188 [email protected]

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A3. Blueberry gummy clinical scripts

PHONE / EMAIL SCRIPT Thank you for your interest in the blueberry gummies for health promotion. ANY INFORMATION COLLECTED WILL BE KEPT CONFIDENTIAL.

The purpose of this research study: We are trying to learn about how compounds in blueberries are absorbed. We will be measuring nutrient-like substances from blueberries in your urine.

What will happen while you are on the study? Two different blueberry confections will be tested in this study. The blueberry confections were developed at OSU in the Department of Food Science and Technology. The confections differ in their blueberry bioactive source. You will be asked to consume ~5 pieces at one time on two occasions.

Specific activities are:  Pre-study Screening Visit You will be asked to come to visit the Clinical Research Center to learn about our study. This will be a brief 10 minute visit. We will describe our study, give you a consent form, and provide you with time to ask any questions. You will be asked to sign or take the consent form home to sign, if you choose to participate. If you decide to participate at that time and are eligible for the study, the study coordinator will provide a study folder.  Enrolled subjects If you decide to volunteer, the study coordinator will schedule your two clinic appointments. All your visits will be in the morning at the OSU Clinical Research Center (CRC). For both visits you will be asked to submit a void/ baseline urine sample before the intervention before your appointment. After you have signed the consent form, each of the two remaining visits should take approximately 15 to 30 minutes. The two visits will likely be close to 30 minutes, because we will ask you to taste several of the confections and complete a sensory analysis. Additionally you will drop off a 24-hour urine collection (collected the day after each visit) to the study coordinator.

Additionally, you will be asked to complete a 3 day diet record (to be completed between the two visits), and complete the daily blueberry diary.

Are you still interested in participating in this study?  Could we have your name, address (home and email), and day/ evening phone numbers?  Potential subjects will be scheduled for their pre-study meeting with a study coordinator at the Clinical Research Center on the OSU Medical Center campus. Directions will be provided.

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To be included in email correspondence: Confidentiality Notice: This e-mail message, from The Ohio State University, Columbus, Ohio, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information. The recipient is responsible to maintain the confidentiality of this information and to use the information only for authorized purposes pursuant to University confidentiality policies. If you are not the intended recipient (or authorized to receive information for the intended recipient), you are hereby notified that any review, use, disclosure, distribution, copying, printing, or action taken in reliance on the contents of this e-mail is strictly prohibited. If you have received this communication in error, please notify us immediately by reply e-mail and destroy all copies of the original message. Thank you.

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A4. Blueberry Gummy Participant Health History Form

Health & Lifestyle Questionnaire

Date______

Name Birth date______Male  Female 

Subject Information

Nationality / Race: ______Occupation: ______Home address: ______Phone No. (H) ______(W) ______Email: ______(C) ______Which is the best way to contact you email and/ or phone? ______Best time to call? ______

Diet / Exercise 1. Do you have any allergies (food or medicines)? Yes  No 

If yes, please specify your allergy and the extent of your allergic reaction: ______

2. Have you ever eaten foods containing blueberries or gelatin?

Yes  No  3. In your regular diet, do you frequently consume red and purple colored fruits or vegetables? Yes  No  If so, how many servings a week? <1  1 to 3  4 to 7  >8 

4. Do you follow a particular diet? Yes  No 

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If yes, please check all that apply. low fat diet  high fiber diet  low carbohydrate diet  no dairy (lactose free)  Atkins diet  macrobiotic  The Zone diet  low sugar diet  40/30/30 diet  low sodium diet  ______(other)  high calorie diet (weight gain) diabetic diet  low calorie diet (weight loss) vegetarian diet  other______

5. Do you exercise regularly?

If yes, please describe your weekly routine (please approximate time spent in each activity) ______

Medical History For male participants please skip to question 4. The first three questions are for female participants. 1. Do you know or suspect that you are currently pregnant, are lactating, or plan to be pregnant during this 16-day study?

Yes  No  2. Are you currently taking any oral contraceptives? Yes  No 

3. Are you menopausal? Yes  When was your last menses? No  Date

4. Are you lactose intolerant? Yes  No 

If yes, how many servings of milk or dairy based products can you tolerate a day?

0 servings  1serving  2 servings  Do you use lactase (Lactaid®)? Yes  No 

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5. Have you taken antibiotics in the last 6 months? Yes  No  If yes, when______how long ______reason ______

6. Have you ever had or currently have any medical problems with the following? If yes, check box Blood (i.e. anemia, bleeding)  Joints (i.e. arthritis)  Chronic Illness (i.e. diabetes)  Kidney  Eating disorders (i.e. bulimia, anorexia)  Large intestine  Swallowing (dysphagia)  Liver (i.e. hepatitis)  Gall Bladder  Small intestine  Immune (i.e. lupus, cancer)  Thyroid or Pituitary 

*If you have checked any of the above boxes, please describe the medical problem. ______7. Have you ever had surgery on any of the following organs? If yes, check box Stomach  Intestines  Thyroid  Gall Bladder  Liver  Pituitary 

8. Do you take prescription medications? Please list with dose taken each day (if known). ______

9. Do you take vitamin or mineral supplements or dietary supplements?

Please list with dose taken each day (if known). ______115

______

10. Do you take any herbal, botanical or “alternative-medicine” preparations? Please list with dose taken each day (if known). ______

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A5. Berry- restricted diet

Name:______Day: ______Date: ______

Berry Restricted Diet: Please try and avoid the following foods. Each of these foods contains ingredients similar to the blueberry confection. If you accidentally eat any of these foods, please circle the food and document approximately how much of the food you consumed.

 Please avoid all berries, including:

Strawberries Lingonberries Acai Blackberries Elderberries Currants Blueberries Cranberries Bilberries Raspberries Cherries Chokeberries

 Please avoid all berry products, including:

Jam or Jelly made from berries Fruit juice from berries (apple and orange juice are OK) Pie and pastries with berry filling

 Other foods to avoid

Red and white wine Pecans Black-eyed peas Walnuts Black beans Pears Radishes Plums Red Cabbage Blood Oranges Eggplant Grapes/raisins (green and red)

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A6. Three Day Dietary Record

Name:______Day / Date:______

*** Start a new page each day. *** Please be as specific as possible. Note any low fat or reduced fat items.

Time Meal Place Eaten Amount Food Description

Continue on back of this page

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*** Please be as specific as possible. Note any low fat or reduced fat items. Time Meal Place Eaten Amount Food Description

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A7. Blueberry gummy clinical sensory questionnaire

18.1 SENSORY EVALUATION OF CONFECTIONS

Thank you for your participation in this multi-disciplinary investigation. We would like to have your opinions and share your experience with consuming the blueberry confections. This information will be used to improve the blueberry confections for future clinical trials and eventually to consumers. These series of sensory test were designed to describe and measure your perception of the 2 different confections. This sensory test will be in three parts and will require 10 to 15 minutes to complete. The instructions for each set of tests are detailed below:

1. Acceptability Test (Likability or palatability): - This test is used to describe the different ways you may have liked or disliked the confections. Starting from left to right, check the box that most appropriately describes your opinion. - Please indicate your ideal gummy candy (for example licorice or gummy bears) in the blank provided AND rate how much you like or dislike your ideal gummy-like candy. Your opinion of our blueberry confections will be compared to your ideal. Your ideal can be different for each of the different measurements. 2. Just About Right Test - This test is used to describe different aspects of the confections by rating the characteristic as being enough or not enough or too much. 3. General Impressions - These questions are to assess your general experience with the confections and foods related to the confections and your fruit consumption.

Please cleanse your mouth with the water cracker provided and rinse your mouth 3 times with water before starting on a new confection sample. Please taste the samples carefully and according to sample number on each page. You may not go back and re-taste the samples once you completed all the questions for that sample.

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Subject ID ______

Acceptability Test

Blueberry Confections CHECK THE BOX THAT BEST DESCRIBES YOUR OPINION OF THE CONFECTION # 1. Please rate your OVERALL LIKING.

Neither Dislike Dislike Dislike Dislike Like Like Like Like Like Nor Extremely Very Much Moderately Slightly Slightly Moderately Very Much Extremely Dislike

2. Please rate the AROMA (smell). Neither Dislike Dislike Dislike Dislike Like Like Like Like Like Nor Extremely Very Much Moderately Slightly Slightly Moderately Very Much Extremely Dislike

3. Please rate the FRUIT FLAVOR (taste). Neither Dislike Dislike Dislike Dislike Like Like Like Like Like Nor Extremely Very Much Moderately Slightly Slightly Moderately Very Much Extremely Dislike

4. Please rate the SWEETNESS.

Neither Dislike Dislike Dislike Dislike Like Like Like Like Like Nor Extremely Very Much Moderately Slightly Slightly Moderately Very Much Extremely Dislike

5. Please rate the TEXTURE (hardness).

Neither Dislike Dislike Dislike Dislike Like Like Like Like Like Nor Extremely Very Much Moderately Slightly Slightly Moderately Very Much Extremely Dislike

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6. Please rate the GRITTINESS (sandiness).

Neither Dislike Dislike Dislike Dislike Like Like Like Like Like Nor Extremely Very Much Moderately Slightly Slightly Moderately Very Much Extremely Dislike

7. Please rate the BITTERNESS. Neither Dislike Dislike Dislike Dislike Like Like Like Like Like Nor Extremely Very Much Moderately Slightly Slightly Moderately Very Much Extremely Dislike

Just About Right Test

Blueberry Confections

CHECK THE APPROPRIATE BOX THAT BEST DESCRIBES YOUR IMPRESSION OF THE CONFECTION # FLAVOR

Fruit

Much Too Too Just About Too Much Too Little Little Right Much Much

Sweet

Sour

Bitter

AROMA Fruit Much Too Too Just About Too Much Too Little Little Right Much Much

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TEXTURE Stickiness

Much Too Just Too Much Too Too Littl About Much Much Little e Right

Firmness

From the above characteristic mentioned above which contributed to your acceptance of the confections?

______From the above characteristic mentioned above which contributed to your dislike of the confections?

______

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General Impressions

Blueberry Confections

To the best of your knowledge from your experience with the confections please complete the following statements. 1. I could consume two confections ______. □not at all □ four times per day □ twice per day □ daily □weekly 2. I could consume four confections everyday for ______. □not at all □ 3 days or less □1 week or more □ 1 month □ 2 months or more What factors (taste, preparation, packaging, etc…) about the confections helped your ability to consume it daily? ______What factors (taste, preparation, packaging, etc…) about the confections hindered your ability to consume it daily? ______What changes need to be made to improve consumption of the confections? ______What are your opinions towards consuming foods with added health benefits such as the blueberry confections? ______

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General Consumer

Blueberry Confections

1. Which statement best describes how you feel about fruit? □Like very much □Like moderately □Neither like or dislike □Dislike moderately □Dislike very much

2. How often do you consume fruit? □Never □Rarely (less than twice a week) □Occasionally (3 to 5 days/week) □Frequently (>7 days/ week)

3. How often do you consume berries or foods containing them? □Never □Rarely (less than twice a week) □Occasionally (3 to 5 days/week) □Frequently (>7 days/ week)

4. Which statement best describes your opinion about blueberries? □Like very much □Like moderately □Neither like or dislike

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□Dislike moderately □Dislike very much 5. Which statement best describes your opinion about candies with fruit? □Like very much □Like moderately □Neither like or dislike □Dislike moderately □Dislike very much

6. How often do you consume candies with fruit? □Never □Rarely (less than twice a week) □Occasionally (3 to 5 days/week) □Frequently (>7 days/ week)

7. Which statement best describes your opinion about candies? □Like very much □Like moderately □Neither like or dislike □Dislike moderately □Dislike very much

8. How often do you consume candies? □Never □Rarely (less than twice a week) □Occasionally (3 to 5 days/week) □Frequently (>7 days/ week)

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A8. Blueberry gummy 24- hour urine instructions 24 Hour Urine Collection Purpose: We are interested in how blueberry phytochemicals from both a blueberry whole food powder and a blueberry extract are absorbed and excreted in the urine. Therefore, we request that you collect all your urine for 24 hours at the beginning and end of the study. Please be sure to stay well hydrated during your urine collection by drinking at least 6 to 8 eight ounce glasses of water, daily.

How to collect: . The 24-hour urine collection should start after providing a spot urine upon arrival and after consuming one serving of blueberry confection on the day of your scheduled visit to the Clinical Research Center. All urine after this time should be collected in the storage container, including all urine of the following morning, until you have collected for the full 24 hours after you consumed the blueberry confections. Make sure to record start and finish time with date. . Collect urine by directly urinating into open urine storage container, or for women into the “hat” provided. Please make sure that all urine is being collected and securely tighten the screw top of urine storage container after every void. . Please do not overfill the urine storage containers. The urine containers should be stored in a safe place so that children will not have access to it. Place in dry, cool, and non-sun-exposed area. Please refrigerate your urine sample or store in a dark cool place. . It is important that you are well hydrated by drinking lots of fluids. . Please bring completed collection with you to your Clinical Research Center appointment.

Note: Please leave the white tablets found in the urine containers. They will dissolve once urine is collected and will preserve the urine during the 24 hour collection.

Please keep urine container away from the reach of children.

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A9. Nutrient consumption breakdown of clinical participants

4000 3500 3000 2500 2000 Intervention 1 1500 Intervention 2

1000 amount food of (g)/ day 500 0 0 1 2 3 4 5 10 11 12 13 14 15 Participant

Figure 25. Total intake of food/ day

5000 4500 4000 3500 3000 2500 Intervention 1 2000 Intervention 2

1500 Calories Calories (kCal)/ day 1000 500 0 0 1 2 3 4 5 10 11 12 13 14 15 Participant

Figure 26. Total calorie intake/ day

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300

250

200

150 Intervention 1

100 Intervention 2 Totalfat (g)/ day

0 0 1 2 3 4 5 10 11 12 13 14 15 Participant

Figure 27. Total fat intake/ day

600

500

400

300 Intervention 1

200 Intervention 2

Totalcarbohydrates (g)/ day 100

0 0 1 2 3 4 5 10 11 12 13 14 15 Participant

Figure 28. Total carbohydrate intake/ day

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200 180 160 140 120 100 Intervention 1 80 Intervention 2

60 Totalprotein (g)/ day 40 20 0 0 1 2 3 4 5 10 11 12 13 14 15 Participant

Figure 29. Total protein intake/ day

30 Intervention 1

20 Intervention 2 Total fiber Totalfiber (g)/ day

0 0 1 2 3 4 5 10 11 12 13 14 15 Participant

Figure 30. Total fiber intake/day

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