Pre-Harvest Elicitation and Post-Harvest Extraction of Phenolic Phytochemicals from Southern Highbush Blueberries

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Pre-Harvest Elicitation and Post-Harvest Extraction of Phenolic Phytochemicals from Southern Highbush Blueberries
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1 online resource (80 p.)
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english
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Buran, Timothy J
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University of Florida
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Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Food Science and Human Nutrition
Committee Chair:
Gu, Liwei
Committee Members:
Williamson, Jeffrey G
Schneider, Keith R
Goodrich, Renee M

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Subjects / Keywords:
aba -- phytonutrients -- resin
Food Science and Human Nutrition -- Dissertations, Academic -- UF
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Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract:
Blueberries contain high levels of phenolic phytochemicals,such as anthocyanins, flavonols, and procyanidins, with many reported health benefits. The objective of this study was to enhance the biosynthesis of phytochemicals and extract phytochemicals from blueberries. The first part of this thesis examined whether a pre-harvest exogenous abscisic application can positively affect fruit quality,antioxidant capacity and phytochemical content of southern high bushblueberries (Vaccinium corymbosum). Abscisicacid (ABA) is a plant growth regulator that has the potential to increase antioxidant capacity and phenolic content of fruits and vegetables. Two varieties, Star and Windsor, were tested with ABA water solutions of three concentrations (0, 200 and 400 ppm) using a randomized complete block design. Results showed that ABA significantly increased the firmness of berries in bothvarieties, suggesting a ripening delay effect. The effect was more pronouncedin Windsor variety as seen by a lower percentage of ripe and smaller sized berries on ABA treated bushes. Overall, ABA delayed the ripening of blueberries, but did not affect total phenolic content, antioxidant capacity,or the content of individual phytochemicals in ripe blueberries. The second part of this thesis investigated the application of ultrasound assisted water extraction in combination with resin adsorption technique to extract and concentrate phenolic phytochemicals from blueberries. The ultrasound assisted hot water method extracted more polyphenols then hot water alone. However, its extraction efficiency was still lower than methanol. Static adsorption tests showed that FPX66 resin had a higher adsorption capacity and a greater desorptionratio than XAD 7HP or XAD 4 resins. XAD 761 and XAD 1180 had the lowestadsorption capacity and desorption ratio. Kinetic adsorption and isotherms tests revealed that FPX 66 had the highest adsorption efficiency and requiredthe shortest time to reach adsorption equilibrium. Dynamic adsorption on a FPX66 resin column demonstrated that phytochemicals in blueberry water extract (WPB)started to break through after 16 bed volumes of extract was loaded. A completed esorption was achieved using 3 bed volumes of ethanol. The blueberry pomace after hot water extraction was extracted with methanol and yielded the methanol-soluble blueberry phytochemicals (MBP). Sugars were not detected in either WBP or MBP. 100 g of fresh blueberries yielded 1.22 g of WBP and 1.39 g of MBP.The recovery rate of total phenolics was 69.5% in WBP and 0.52% in MBP.   In short, pre-harvest application of abscisic acid was ineffective in enhancing the phytochemical biosynthesis in blueberries. An extraction and concentration method wasdeveloped to successfully produce concentrated sugar-free phytochemicalextracts from blueberries. These extracts were suitable for use as dietarysupplements for people with glucose intolerance or diabetes.
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In the series University of Florida Digital Collections.
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Includes vita.
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by Timothy J Buran.
Thesis:
Thesis (M.S.)--University of Florida, 2012.
Local:
Adviser: Gu, Liwei.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-08-31

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1 PRE HARVEST EL ICITATION AND POST HARVEST EXTRACTION OF PHENOLIC PHYTOCHEMICALS FROM SOUTHERN HIGHBUSH BLUEBERRIES By TIMOTHY J. BURAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DE GREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012

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2 2012 Timothy J. Buran

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3 To my parents, John and Sue, and my brother, Steven; You have always been there to break my fall with every giant leap. The following thesis is a result of one of those

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4 ACKNOWLEDGMENTS I thank my parents, John and Sue, and my brother, Steven for their continuous support throughout my personal and professional career. I also thank Dr. Gu for taking me on as

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 10 1 LITERATURE REVIEW ................................ ................................ .......................... 12 Introduction ................................ ................................ ................................ ............. 12 Phytochemical compos ition in blueberries ................................ .............................. 13 Health Benefits of Blueberry Phytochemicals ................................ ......................... 14 Diabetes in the U.S. ................................ ................................ ................................ 15 How Blueberries Can Be Used to Help Those with Diabetes ................................ .. 16 Latest Research ................................ ................................ ................................ ...... 19 Conclusion and Research Objectives ................................ ................................ ..... 21 2 EFFECTS OF EXOGENOUS ABS CISIC ACID ON FRUIT QUALITY, ANTIOXIDANT CAPACITI ES, AND PHYTOCHEMICA L CONTENTS OF SOUTHERN HIGH BUSH B LUEBERRIES ................................ ............................. 23 Introduction ................................ ................................ ................................ ............. 23 Materials and Method s ................................ ................................ ............................ 24 Chemical Reagents. ................................ ................................ ......................... 24 ABA Applications. ................................ ................................ ............................. 24 Polyphenol Extraction. ................................ ................................ ...................... 25 Folin Ciocalteu Assay. ................................ ................................ ...................... 25 Oxygen Rad ical Absorbance Capacity (ORAC FL ) Assay. ................................ 26 DPPH Assay. ................................ ................................ ................................ .... 26 Texture and Color Analysis. ................................ ................................ ............. 27 HPLC ESI MS n Analyses of Phytochemicals. ................................ .................. 28 Statistical Analyses. ................................ ................................ ......................... 29 Results ................................ ................................ ................................ .................... 29 Berry Quality. ................................ ................................ ................................ .... 29 Total Phenolic Content and Antioxidant Capacity. ................................ ............ 30 Phytochemical Identification and Quantification. ................................ .............. 31 Discussion ................................ ................................ ................................ .............. 31 Conclusions ................................ ................................ ................................ ............ 34

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6 3 EXTRACTION AND CONCE NTRATION OF PHYTOCHE MICALS FROM BLUEBERRIES USING UL TRASOUND ASSISTED EX TRACTION AND RESIN ADSORPTION ................................ ................................ ................................ ........ 40 Introduction ................................ ................................ ................................ ............. 40 Materials and Methods ................................ ................................ ............................ 41 Chemicals ................................ ................................ ................................ ......... 41 Pre Treatment of Resins ................................ ................................ .................. 41 Extraction of Phytochemicals from Blueberries ................................ ................ 41 Static Adsorption/Desorption Testing ................................ ............................... 42 Adsorption Kinetics ................................ ................................ .................... 44 Adsorption I sotherms and Thermodynamics ................................ .............. 44 Dynamic Adsorption/Desorption Testing ................................ .......................... 45 Folin Ciocalteu Assay ................................ ................................ ....................... 45 Total Anthocyanin Content ................................ ................................ ............... 46 Total Procyanidin Content ................................ ................................ ................ 46 Oxygen Radical Absorbance Capacity (ORAC FL ) Assay ................................ .. 46 HPLC Analyses of Phytochemicals and Sugars ................................ ............... 47 Statistical Analyses ................................ ................................ .......................... 48 Results and Discussion ................................ ................................ ........................... 48 Extraction of Polyphenols Using Power Ultrasound ................................ ......... 48 Static Adsorption and Desorption ................................ ................................ ..... 49 Adsorption kinetics ................................ ................................ ..................... 51 Adsorption isotherms and thermodynamics of FPX 66 resin ...................... 52 Dynamic Adsorption/Desorption on FPX 66 Resin ................................ ........... 53 Phytochemical and Sugar Composition in Extracts ................................ .......... 54 Yield and Recovery of Polyphenols ................................ ................................ .. 54 Conclusion ................................ ................................ ................................ .............. 55 4 FINAL CONCLUSIONS ................................ ................................ .......................... 73 LIST OF REFERENCES ................................ ................................ ............................... 74 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 80

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7 LIST OF TABLES Table page 2 1 Fruit quality of blueberries as affected by ABA treatments ................................ 35 2 2 T otal phenolic content and antioxidant capacities of ripe blueberries as affected by ABA treatment ................................ ................................ .................. 36 2 3 Content of anthocyan ins, flavonols, and chlorogenic acid in ripe berries of Star variety as affected by ABA treatment ................................ .......................... 37 2 4 Content of anthocyanins, flavonols, and chlorogenic acid in ripe berries of Windsor variety as affected by ABA treatments ................................ .................. 38 3 1 Chemical and physical proper ties of resins ................................ ........................ 56 3 2 Extraction efficiency of different solvents under different extraction conditions .. 57 3 3 Pseudo first and second order rate constants of resins calculated on the basis of total phenolics, total anthocyanins and total procyanidins ..................... 58 3 4 Langmuir and Freundlich equation constants on Amberlite FPX 66 resin .......... 59 3 5 Content of individual phenolic compounds in fresh blueberries, WBP, and MBP ................................ ................................ ................................ .................... 60 3 6 Yield, ORAC, total phenolic content in fresh blueberries, WBP and MBP .......... 61

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8 LIST OF FIGURES Figure page 2 1 HPLC chromatograms phenolic compounds in blueberries of Star variety. ....... 39 3 1 Flow chart for the extraction and concentration of blueberry phytochemicals. .... 62 3 2 Static adsorption and desorpti on results based on total anthocyanins. .............. 63 3 3 static adsorption and desorption results based on total phenolics. ..................... 64 3 4 Static adsorption and desorption results based on total procyanidins. ............... 65 3 5 Kinetic curves of total anthocyanins on Amberlite resin FPX 66, XAD 4, and XAD 7HP.. ................................ ................................ ................................ .......... 66 3 6 Kinetic curves of total phenolics on Amberlite resin FPX 66, XAD 4, and XAD 7HP. ................................ ................................ ................................ ................... 67 3 7 Kinetic curves of total procyanidins on Amberlite resin FPX 66, XAD 4, and XAD 7HP. ................................ ................................ ................................ ........... 68 3 8 Adsorption isotherms and thermodynamics of total anthocyanins on Amberlite resin FPX 66 ................................ ................................ ....................... 69 3 9 Adsorption isotherms and thermodynamics of total phenolics on Amberlite resi n FPX 66. ................................ ................................ ................................ ...... 70 3 10 Dynamic adsorption curves of total anthocyanins on Amberlite resin FPX 66 at different flow rates ................................ ................................ ......................... 71 3 11 Dynamic desorption curves of total anthocyanins on Amberlite resin FPX 66 at different flow rates ................................ ................................ ......................... 71 3 12 HPLC chromatogram of sugars in hot water extract using reflective index detection. Peak 1 and 2 were fructose and glucose, respectively. ..................... 72 3 13 HPLC chromatogram of anthocyanins in sugar free resin produced blueberry extract at 520 nm detection.. ................................ ................................ .............. 72

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9 LIST OF ABBREVIATION S ABA Abs cisic Acid ORAC Oxygen Radical Absorbance Capacity MPB Methanol soluble blueberry phytochemicals WPB Water soluble blueberry phytochemicals

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10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science PRE HARVEST ELICITATION AND POST HARVEST EXTRACTION OF PHENOLIC PHYTOCHEMICALS FROM SOUTHERN HIGHBUSH BLUEBERRIES By Timothy J. Buran August 2012 Chair: Liwei Gu Major: Food Science and Human Nutrition Blueberries contain high level s of phenolic phytochemicals, such as anthocyanins, flavonols, and procyanidins, with many reported health benefit s The objective of this study was to enhance the biosynthesis of phytochemicals and extract phytochemicals from blueberries. The first part of this thesis examine d whether a pre harvest e xogenous abscisic application can positively affect fruit quality, antioxidant capacity and phytochemical content of southern high bush blueberries ( Vaccinium corymbosum ). Abscisic acid (ABA) is a plant growth r egulator that has the potential to increase antioxidant capacity and phenolic content of fruits and vegetables. Two varieties, Star and Windsor, were tested with ABA water solutions of three concentrations (0, 200 and 400 ppm) using a randomized complete b lock design. Results showed that ABA significantly increased the firmness of berries in both varieties, suggesting a ripening delay effect. The effect was more pronounced in Windsor variety as seen by a lower percentage of ripe and smaller sized berries on ABA treated bushes. Overall, ABA delayed the ripening of blueberries, but did not affect total phenolic content, antioxidant capacity, or the content of individual phytochemicals in ripe blueberries

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11 The second part of this thesis investigated the applica tion of ultrasound assisted water extraction in combination with resin adsorption technique to extract and concentrate phenolic phytochemicals from blueberries The u ltrasound assisted hot water method extracted more polyphenols then hot water alone. Howev er, its extraction efficiency was still lower than methanol. Static adsorption tests showed that FPX66 resin had a high er adsorption capacity and a greater desorption ratio than XAD 7HP or XAD 4 resins XAD 761 and XAD 1180 had the lowest adsorption capacity and desorption ratio. Kinetic adsorption and isotherms tests revealed that FPX 66 had the highest adsorption efficiency and required the shortest time to reach adsorption equilibrium Dynamic adsorption on a FPX 66 resin column demonstrated that phytochemicals in blueberry water extract (WPB) started to break through after 16 bed volumes of extract was loaded. A complete desorption was achieved using 3 bed volumes of ethanol. The blueberry pomace after hot water extraction was extracted with metha nol and yielded the methanol soluble blueberry phytochemicals (MBP). Sugars were not detected in either WBP or M BP. 100 g of fresh blueberries yielded 1.22 g of WBP and 1.39 g of MBP. The recovery rate of total phenolics was 69.5 % in WBP and 0. 52 % in MBP. In short, p re harvest application of abscisic acid was ineffective in enhanc ing the phytochemical biosynthesis in blueberries. An extraction and concentration method was developed to successfully produce concentrated sugar free phytochemical extracts fro m blueberries. These extracts were suitable for use as dietary supplement s for people with glucose intolerance or diabetes.

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12 CHAPTER 1 LITERATURE REVIEW Introduction Fruits and vegetables are known to help with disease prevention and are beneficial to maintaining a healthy lifestyle. Fruits with a high content of phytochemicals have been researched in order to determine their potential health benefits. Phytochemicals are known to assist in the prevention of many different diseases includi ng cancer, cardiovascular disease and diabetes. Phytochemicals are also known to help in the scavenging capabilities of oxidative free radicals, which are known to lead to the development of several chronic diseases (Hooper & Cassidy, 2006) Currently, research is focusing on the effect that berries, specifically blueberries have on the human diet. Berries in general are low in calories and contain a number of antioxidants. They include v itamins C and E and other important micronutrients such as folic acid, calcium, alpha and beta carotene, and lutein (Basu, Rhone & Lyons, 2010) Blueberries have been shown to contain these health promoting effects as well as having one of the highest antioxidant capacities of all fruits and vegetables commonly consumed (Harborne & Williams, 2000) This has led to numerous research s tudies investigating the effect that b lueberries have on the human health system. According to the US Highbush Blueberry Council, the average fresh berry contains around 10.65 g of sugar per 100 g of blueberry (USHBC, 2012) There are over 8.3 million people in the US with sugar li mitations from diabetes (CDC, 2011) who would not b e able to enjoy the numerous health benefits associated with the consumption of fresh blueberries. C urrently, research is being conducted to determine the effect that

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13 blueberries have on the prevention of illnesses stemming from diabetes as well as method s to create all natural, calorie free, sugar free extracts from fresh blueberries. Phytochemical composition in blueberries Blueberries contain a variety of phytochemicals including several beneficial nutritional compounds such as flavonoids and p henolic a cids. These phenolic compounds are shown to have numerous biological effects which include antioxidant and ant i carcinogenic properties Blueberries have been identified to have several different flav o noids ; t he most commonly found are anth ocyanin s and flavonols (Cho, Howard, Prior & Clark, 2004) Research has shown that consumption of fl av o noids ha s preventative effects against heart di sease, stroke, and lung cancer (Zheng & Wang, 2002) Flavonoids are polyphenolic compounds which have a C15 base skeleton and represent the majority of secondary metabolites found in plants. Flavonoids n aturally occur in plants as a way to prevent oxidative stress. The oxidative stress is caused by the promotion of several free radicals. Free radicals can form throughout the environment and are necessary in some situations. Free radical formation can occu r either from within our own body through intracellular mechanisms or through external environmental conditions. Internally, free radical formation is believed to be caused by autoxidation, along with the reduction of smaller molecules such as thiols and f lavins. Externally, free radical formation can occur through exposure to environmental pollution, smoking, organic solvents, and pesticides (Freeman & Crapo, 1982) The formation of free radicals is unavoidable but their effects can be reduced by diet choices Research has led us to believe that the cons umption of foods rich in f lavonoids may lead to the reduction of t hese chronic diseases Since f lavonoids

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14 are rich in antioxidants, their free radical scavenging ability can help reduce the damage caused by the production or consumpti on of free radicals in the body (Cho et al., 2004) A nth ocyanins are commonly found in many produce products with over 600 known na turally occurring anth ocyanins identified (Andersen & Markham, 2006) In the food industry, anth ocyanins have become a popular way for companies to add an using them as food colorants Also, the numerous health benefits including prevention of heart disease and cancer, are believed to be caused by the anth ocyanins rich antioxidant capacity (Macheix, Fleuriet & Billot, 1990) Phenolic a cids are known to be h igh in antioxidant capacity like their counterparts, f lavonoids (Rice Evans & Miller, 1996) They are found in high numbers in blueberries and aid in the numerous heath benefits that blueberry phytochemicals have to offer. Diff erent p henolic a cids are found in different varieties of blueberr ies Also, depending on the time of year and the age of the blueberry, analysis will show different concentrations of p heno lic a cids. Some common phenolic a cids in blueberries are chlorogenic acid gallic acid, caffeic acid and ferulic acid ; a ll of which have the potential to be free radical scavengers once ingested in the body (Sellappan, Akoh & Krewer, 2002) He alth Benefits of B lueberr y P hytochemicals There are several health benefits of including blueberries in the diet. Aside from their extremely high antioxidant capabilities, they also contain additional nutrients that aid in maintaining a healthy immune s yst em. According to the USDA a serving of blueberries (184 g) contains 14 mg or about 25% of your daily requirements for v itamin C. Blueberries are also a good source of fiber. Besides regulating a healthy immune system, fiber has been shown to improve heart health by maintaining good cholesterol

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15 (USDA, 2006) According to the National Academy of Sciences, blueberries are also an excellent source of Manganese which is known to improve the metabolism of proteins, fats and carbohydrates (IOM, 2001) There has also been some research done on the effect that phytochemicals have on the reduction of coronary heart disease as well as a reduction in the prevalence of cancer (Hertog M. & et al., 1995) The ingestion of flavanols has been known to have some anti carcinogenic and anti atherogenic effects. For ex ample, research has shown that delphinidin and malvidin glycosides have been known to cause cell death in leukemia HL60 cells as well as show a reduction in HCT116 cells which are known to cause colon cancer (Youdim, McDonald, Kalt & Joseph, 2002) Although blueberries have been shown to help prevent several diseases (Hooper et al., 2006) research linking the health benefits of blueberries to diabetes has been limited. Diabetes affects over 143 million individuals worldwide and it is estimated by the year 2030, there will be over 200 million individuals diagnosed with t ype 2 d iabetes. Diabetes itself is associated with the damaging effects caused by oxidative stress. The most likely source of oxidative stress in diabetics is hypoglycemia (low blood sugar) and dyslipidemia (high blood cholesterol). Diabetes in the U S According to the 2011 National Diabetes Fact Sheet collected by the Center s f or Disease Control and Prevention (CDC) over 25.8 million children and adults have been diagnosed with diabetes in this country alone. That is almost 8 .3 % of the total US p opulation. Th is does not include the over 57 million individuals that are pre diabetic The m ajority of these individuals is overweigh t and can prevent becoming diabetic by

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16 changing their lifestyle The most common diabet ic patients are adults aged 20 and a bove with the majority, about 27 %, being over the age of 60. According to the same survey, d iabetes was the 7 th leading cause of death. The se deaths stem from the disease itself or complications caused by the disease. Research has shown that diabetic individuals are 2 to 4 times more likely to suffer a stroke as well as develop heart disease. I ndividuals with diabetes reported elevated blood pressure ( greater than or equal to 130/80) and/or were being prescribed medication for hypertension. Another complication related to diabetes is blindness d iabetic r etinopathy has been shown to create 12,000 24,000 additional cases a year. Also, b etween 60 70 % of patients with diabetes are shown to have mild to severe damage to their nervous system and over 60% of diagnosed individuals had to have some form of non traumatic lower limb amputations. D iabetes can also be a f inancial burden as well. In 2011 total costs associated for the patients diagnosed with the disease were over $170 b illion. This was not including the over $ 40 b illion in cost s associated with undiagnosed or pre diabetic patient complications These costs are about 2.3 times higher than someone who is not diabetic (CDC 2011) How B lueberries C an Be Used to Help T hose with Diabetes The most com mon form of diabetes is type 2 d iabetes which is caused by the development of insulin resistance. Insulin is produced in the pancreas. This insulin secretion is necessary to p revent fat breakdown in the adipose tissue and to promote the storage of glucose in the liver. Adipose tissue develops from the over consumption of fats in the diet and is stored the stored form for later energy use. A breakdown of this fat will prevent the proper storage of energy. Those with insulin resistant type 2 d iabetes

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17 will usually have an increase of circulating fatty acids and increased glucose in the liver (Shulman, 2000) D i abetes itself is associated with the damaging effects that oxidative stress can cause. The most like ly source of oxidative stress is hypoglycemia (low blood sugar) and dyslipidemia (high blood cholesterol). Both of these conditions aid in the development of type 2 diabetes by inducing inflammatory immune responses. These responses occur when a person bec ome s ill and free radical formation occurs The creation of these unnecessary free radicals is what is believed to be the cause of some of the complications associated with type 2 diabetes (Baynes & Th orpe, 1999; Pickup, 2004) These free radicals have been known to be reduced by the addition of antioxidants in the diet. Antioxidant rich foods are one s that contain high amounts of f lavonoids, carotenoids and ascorbic acid. These ingredients are comm only found in most berries and are found in high concentrations in blueberries. These components are known to inhibit free radicals by several different reaction enzymes (Manach, Mazur & Scalbert, 2005) Both type 1 and t ype 2 dia betes are known to have a negative effect on pancreatic B C ells. These cells are very important as they control the creation and release of insulin into the body from the pancreas. Diabetes affects the ability of these B Cells to properly function, by gradual reduction (t ype 2) or co mpletely destroying them (t ype 1). A study looking at the anti diabetic properties of Canadian lowbush blueberry found that blueberry extracts were shown to have positive effects when it came to altering these B Cells. R esearch shows that blueberry fruit extracts had a positive effect on the proliferation of these B Cells. These results lead to the belief that t he extract could help

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18 prevent the deterioration of these B Cells resulting in reduced cell de struction in advan ced cases of t ype 2 diabetes (Martineau et al., 2006b) Glucose stimulated insulin secretion (GSIS) in pancreatic B Cells is important for regulating the blood sugar levels in the human body. GSI S is dependent on a series of factors such as coordinated glucose uptake and oxidative metabolism (Norlin, Ahlgren & Edlund, 2005) In a diabetic patient GSIS is impaired and depending on the type of diabetes, will either secrete too much or not enough insulin into the blood stream. Studies showed that the leaf and stem extracts of blueberries have positive effects for diabetic patients by improving GSIS. Stem and leaf extracts showed an increase in maximal insulin secretion as well as a leftward shift in the glucose insulin dose response curve. These are two very important factors experts review when determining the effectiveness of almost any medication prescribed to diabetic patients. By increasing insulin secretion along with shifting the dose respo nse curve, patients will begin to see a decrease in the chances of inducing hypoglycemia, one of the largest concerns for diabetic patients (Bailey, 1999) Diabetic patients have several complications relating to their vision. In some recent studies, blueberries have been sh own to help increase vision in impaired individuals. This is possible because blueberries help in the generation of retinal pigments which has led to the improvement of night vision. Also blueberries allow for increased circulation in the capillaries of the retina therefore improving vision. In a study by Head in 2001 found that subje cting individuals to a diet in anth ocyanins individuals on the diet that included anth ocyanins had an increase in their ability to see visual purple. This is

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19 an important light pigment as it helps convert the light that is seen i nto signals the brain can interpret (Camire, 2000; Head, 2001) Latest R esearch D iabetes is associated with high occurrences of oxidative stress specifically due to the hyperglycemia and hyperlipidemia. These lead to the body inducing an inflammatory immune response as well as oxidative stress. When oxidative stress occurs, free radical production ensues which is believed to be one of the factors leading to complications relating to adult onset type 2 diabetes (Baynes et al., 1999; Pickup, 2004) Recently metabolites from blueberry polyphenols produced by gut flora have been shown to decrease this inflammation in vitro which was measured by prostanoid production. When exposed to these beneficial metabolites a positive immune response was shown in human endothelial cells The anthocyanin metabolites reduced TNF alpha expression and oxidative damage (Boyle & Leone, 2008; Hou, Yanagita, Uto, Masuzaki & Fujii, 2005; Youdim et al., 2002) In a study by Vuong and others (2009) the researchers looked at the effects of blueberry juice in vivo comparing biotransformed blueberry juice to a non blueberry juice. Biotransformed juice was prepared by taking fresh low bush blueberries and inoculating the samples with S erratia accinii bacteria The samples were allowed to ferment for four days and then were put through a sanitation and purification method. The non blueberry juice was a mixture of water, glucose, fructose maltose, and sucrose matching the 7.9% w/w volume of known sugar contained in the blueberry juice. T he study showed several prom ising results including that the blueberry j uice diet significantly reduced the cumulative food intake of the mice when compared to the control and non blueberry juice group s All of the mice in the diabeti c control group and

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20 the non blueberry j uice group showed high non fasting glycemia levels (at or above 20mM). Ho wever, only 20% of mice on the blueberry j uice diet showed non fasting glycemia levels at or above 20 mM suggesting that the biotransformed blueberry juice may positively impact non fasting glycemia levels in diabetic patients (Vuong et al., 200 9) Several studies and models have been done investigating the effects of antioxidant rich fruits on diabetic patients. A 2009 study by Grace and others investigated th e effect of blueberry in vivo C57b1/6J diabetes induced mice were used for the tes ting. Labrasol an approved drug carrier, was combined with two different type s of extracts; a phenolic rich extract as well as a n anthocyanin enriched fraction. Researchers found that when phenolic rich blueberry fractions were administered blood g lucose levels decreased by 33%. The anthocyanin rich fractions did even better, decreasing blood glucose levels by 51% when compared to the control (Grace et al., 2009) Research focused in the area of anthocyanin rich fruit is not just limited to blue berries. In a study by Tsuda and others in 2003 anthocyanins from purple corn were investigated to see if there was any effect on obesity on the same mice model. Results showed that mice fed a high fat diet combined with concentrated anthocyanins from purple corn, showed a decrease in serum levels of glucose, insulin, and leptin concentrations when c ompared to the high fat control diet. The same group later showed gene expression of adiponectin was shown to be up regulated in white adipose tissue for those mice fed a diet with added anthocyanins. This led researchers to believe that there was a possib ility that anthocyanins from purple corn could in fact

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21 reduce the risks of obesity and diabetes (Tsuda, Horio, Uchida, Aoki & Osawa, 2003; Tsuda et al., 2004) This idea led to research investigating the effects of whole blueberries along with a concentrated anthocyanin extract on obesity and diabetes. The study was set up very similar to the above study as they also used C57b1/6J mice and fed them high fat diets. Results indicated that those mice on a low fat die t saw no significant change in weight gain or b ody fat. However, mice on a whole blueberry hig h fat diet showed a n increase in body weight body fat and epididymal fat weights when compared to the high fat control diet Anthocyanins fed as a whole blueberry showed negative effects H owever, when an extraction and concentration method for anthocyanins was employed and pl aced in the diet, a reduction in obesity was noted (Prior, Wu, Gu, Hager, Hager & Howard, 2008b) Conclusion and Research O bjectives Blueberries have been deemed the king of superfruits With their numero us health benefits and disease preventing abilities, it is clear why the research world has a vested interest in blueberries. As previously mentioned, blueberries are full of phytochemicals with high amounts of anthocyanins, flavonoids, and phenolic a cids. These components along with the numerous other nutrients contribute to the Blueberries are not a perfect food by any means. Currently, natural sugars prevent those that are sensitive to sugar ( i.e. diabetic pat ients ) from enjoying the benefits of blueberries. It is important to note, blueberries are kn own to have numerous anti diabetic effects and could help reduce the complications diabet es has on the body (Martineau et al., 2006a) With over 27 % of this country either diabetic or pre diabetic,

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22 there is a large population that cannot enjoy the benefits this fruit has to offer Currently food chemistry research is focusing on developing a method to extract and purify phytochemicals from blueberries while excluding the sugars. This research would a llow diabetic patients to receive the anti diabetic effects without the consumption of sugar. With new health advances occurring daily and a rise in popularity of natural food s it is no surprise that superfruits such as blueberries are being studied. Alt hough not much research has been completed on the health benefits of blueberries specifically, the curiosity is there and the avenues for exploration are immense. There were two research objectives for this thesis: 1. T o determine if an exogenous treatment of a bscisic a cid affects phytochemical content in two varieties of southern high bush blueberries 2. To use p ower ultrasound assisted hot water extraction along with resin adsorption technology to produ ce sugar free blueberry extracts.

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23 CHAPTER 2 EFFECTS OF EXOGENOUS ABSCISIC ACID ON FRU IT QUALITY, ANTIOXID ANT CAPACITIES, AND PHYT OCHEMICAL CONTENTS O F SOUTHERN HIGH BUSH BLUEBERRIES Introduction Blueberries have a high content of phytochemicals, including anthocyanins, flavonols, and phenolic acids. They are known to prevent cancer, cardiovascular diseases, diabetes, and scavenge oxidative free radicals (Hooper et al., 2006) Abscisic affects plant growth in numerous ways including seed maturity or dormancy, as well as vegetative tissue reactions when placed under environmental stress (Baumann, 2010) Pr evious studies have shown that exogenous ABA application affects growth and phytochemical biosynthesis in several fruits and vegetables. The capacity of ABA to increase the phytochemical content is of particular interest for food and nutritional sciences a s it increases the nutritional v alue of fruits and vegetables. In a recent study involving greenhouse red and green leaf lettuces, exogenous ABA application significantly increased the anthocyanin content in red leaf lettuce along with the contents of chlo rophyll b and total carotenoids in green leaf lettuces when compared to the control (Li, Zhao, Sandhu & Gu, 2010) Exogenous ABA was found t o stimulate anthocyanin biosynthesis and increase the content of phenolic compounds in Noble muscadine grapes, but not in the Alachua muscadine grapes (Sandhu, Gray, Lu & Gu, 2011) The effect of ABA on phytochemical rich berries such as southern high bush blueberries is not known It was hypothesized that an exogenous application of ABA on blueberries may have a positive impact on their antioxidant capacity as well as their phytochemical content. The present study was designed to test these hypotheses by

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24 evaluating the effects of the exoge nous ABA on the yield, quality, antioxidant capacities, and phytochemical contents of southern high bush blueberries ( Vaccinium corymbosum ). M aterials and M ethods Chemical Reagents azotis(2 amidinopropane)) was a product of Wako Chemicals Inc. (Bellwood, RI). 2,2 Diphenyl 1 picrylhydrazyl (DPPH), Trolox (6 hydroxy 2,5,7,8 tetramethylchroman 2 carboxylic acid), and cyanidin 3 rutinoside were purchased from Sigma Aldrich (St. Louis, MO). Folin Ciocalteu reagent, Tween 20, quercetin, chloroge nic acid, and other chemicals were products of Fisher Scientific (Pittsburg, PA). Standards of the 3 O glucosides of pelargonidin, cyanidin, peonidin, delphinidin, petunidin, and malvidin were purchased from Polyphenols Laboratories (Sandnes, Norway). T he S abscisic acid was received as a gift from Valent BioSciences Corporation (Libertyville, IL). ABA Applications. ABA treatments on blueberries were conducted in an experimental orchard in Citra, Florida. Five year old Star and Windsor varieties of sou thern high bush blueberries of the same size and growing conditions were used. The experiment was arranged in a randomized complete block design with 12 replications. There were three plants per block that were given an ABA treatment in three concentration s [0 (control), 200, and 400 ppm]. ABA was dissolved in water that contained 315 L/L of Tween 20 as a wetting agent. Control solution contained water with Tween 20. ABA or control solutions were sprayed on berries until run off using a garden sprayer. Abo ut half of the leaves were also wetted after the spray. Bushes used in this experiment were separated

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25 by at least one guard plant. The first spray was carried out on April 5, 2010 when the was perform ed on April 28, 2010 when the berries were in the same growing stage with 5 10% of the berries being green with a pink hue. The third spray followed on May 6, 2010 when 40 70% of berries on the bushes showed a similar pink hue. The first sampli ng was done on May 6, 2010 before the third ABA spray. Eight clusters of berries were randomly picked from each bush. Half of the clusters were picked from one side of the row and the other half were from the opposite side. The second sampling was done on May 12, 2010 using the same sampling protocol. The total numbers of berries, number of ripe and unripe berries on a cluster were recorded. Ripe and unripe berries were separated and analyzed for weight, color, and firmness. Ripe berries were freeze dried a nd analyzed for total phenolic content, antioxidant capacity, and phytochemical content. Polyphenol Extraction. Blueberries were freeze dried and extracted one month later using the following method. Freeze dried ripe blueberries (0.25 g) were weighed in to 30 mL screw capped glass tubes and 20 mL methanol: water: acetic acid (85:15:0.5, v/v/v) was added as an extraction solvent. The tubes were vortexed for 30 s ec. and sonicated for 5 min, and kept at room temperature in dark for 20 min an d vortexed again for another 30 s ec The tubes were centrifuged at 3 000 rpm for 10 min. Supernatants were decanted out and kept at 20 o C until further analysis. Folin Ciocalteu Assay. Total phenolic content was determined by the Folin Ciocalteu assay. Blueberry extracts were mixed with diluted Folin Ciocalteu reagent and 15% sodium carbonate. Absorbance at 765 nm was measured on a SPECTRAmax 190 microplate reader

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26 (Molecular Devices, Sunnyvale, CA) after 30 min of incubation at room temperature. Gallic acid was used to ge nerate a standard curve. Results of total phenolic content for blueberries were expressed as milligram gallic acid equivalent per gram of fresh blueberry samples (mg GAE/g). Oxygen Radical Absorbance Capacity (ORAC FL ) Assay. Blueberry extracts were incub ated with fluorescein as a free radical probe and AAPH as a free radical generator (Prior et al., 2003) The kinetics of fluorescein degradation was read on a Spectra XMS Gemini microplate reader (Molecular Devices, Sunnyvale, CA). 6 Hydroxy 2,5,7,8 tetramethylchroman 2 carboxylic acid (Trolox) was used to generate a standard curve. The results of the antioxidant capacity of blueberries were expressed as mol Trolox equival ent per gram of fresh blueberry samples (mol TE/g). DPPH Assay. The DPPH (2,2 diphenyl 1 picrylhydrazyl ) scavenging activities of freeze dried blueberries were measured using a published method (Brand Williams, Cuvelier & Berset, 1995) Twenty milligrams of DPPH was dissolved into 100 mL of methanol to make a DPPH stock solution DPPH working solution was freshly prepared by mixing 3.5 mL DPPH stock solution and 6.5 ml methanol. Absorbance at 515 nm was measured on a SPECTRAmax 190 microplate reader (Molecular Devices, Sunnyvale, CA). The initial absorbance of DPPH working soluti on was between 0.9 1.0. The blueberry extracts (50 L) were added to 950 L DPPH working solution and samples were kept at room temperature in dark for 60 min. Trolox solutions from 100 1000 M were added to DPPH working solution as standards. The results of the DPPH

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27 scavenging activity of blueberries were expressed as mol Trolox equivalent per gram of fresh blueberry sample (mol TE/g). Texture and Color Analysis. Textural analysis of blueberries was performed using the TA.XT Plus Texture Analyzer (Text ure Technologies Corp., Scarsdale, NY). Blueberries were compressed using a flat probe from 25 mm distance at a speed of 10 mm/s with a 1 g contact force. Maximum peak force was obtained from five readings of duplicated samples. Color analysis was run usi ng a machine vision system consisting of a light box (Luzuriaga, Balaban & Yeralan, 1997) a Nikon D200 digital color camera and a Nikon DX 18 200mm VR II Lens (Nikon Corp., Tokyo, Japan) connected to a computer through a USB cable, using Lens Eye Software (Gainesville, F L ). The light box used 2 fluorescent light bulbs (Lumichrome F15W1XX, color temperature = 6500 K, color retention index= 98, Lumiram, Larchmont, N Y ) emulating the D65 illumination (natural daylight at noon). Diffuse light inside the box was obtained by using a Polycast acrylic nr 2447 plastic sheet (Faulkner Plastics, Gainesville, F L ) between the fluorescent bulbs and the sa mple space. The camera settings were as followed; exposure mode: f/8, aperture, shutter speed: 0.5 sec: 0 exposure value, exposure compensation: 0 exposure value, sensitivity: 250 ISO, color temperature: 5600 K, hue adjustment: 6 o Color analysis was per formed using Lens Eye software (Engineering & Cyber S olutions Inc., Gainesville, F L ) using Visual Basic for Windows (Microsoft, Redmond, W A ). The blueberry samples were placed at the bottom of the light box and a picture was captured from the digital camer a set on a tripod facing the samples at the bottom of the light box. The captured images (1000 x 669 pixels) taken with the MV system were calibrated with a Labsphere (North Sutton, N H ) standard blue plate (L = 58.24, a = 4.74, b = 42.44). The images

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28 were 24 bit color, meaning that in the r ed, g reen, b lue (RGB) color space, each color axis was represented by 8 bits or 2 8 = 256 different values. Using the LensEye software, first the RGB values of every pixel of a sample image were read, then this color information was converted to the L*, a*, and b* values, and averaged for each blueberry sample. A threshold was establishe d to determine the ripeness stage using the average b* values of each sample. HPLC ESI MS n Analyses of Phytochemicals. An Agilent 1200 HPLC system consisting of an autosampler, a binary pump, a column compartment, a diode array detector and a fluorescent detector (Agilent Technologies, Palo Alto, CA) was interfaced to a HCT ion trap mass spectrometer (Bruker Daltonics, Billerica, MA). Samples were centrifuged at 17 000 rpm for 10 min using an AccuSpin Micro 17 Centrifuge System (Fisher Scientific, Pittsbur g, PA). The supernatant was removed and 10L of sample was injected. An Agilent Zorbax Stablebond SB Technologies, Palo Alto, CA) was used for the separation of phytochemicals. The binary mobile phas e consisted of (A) formic acid: water (5:95 v/v) and (B) methanol. For the analysis of flavonols, chlorogenic acid derivatives and anthocyanins, a 70 min gradient was used and adapted from (Cho et al., 2004) The gradient is described as follows: 0 min 5% B, 2 min 5% B, 10 min 20% B, 15 min 20% B, 30 min 25% B, 35 min 25% B, 50 min 33% B, 55 min 40% B, 60 min 60% B, 65 min 70% B, 7 0 min 5% B isocratic; followed by 5 min of re equilibration of the column before the next run. The detection wavelength was 520 nm for anthocyanins, and 360 nm for chlorogenic acid and flavonols. Electrospray ionization in alternating mode was performed us ing nebulizer 30 psi, drying gas 11 L/min, drying temperature 300 o C, and capillary of 4000 V allowing

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29 compounds to be detected in both positive and negative mode in the same run. The full scan mass spectra of the anthocyanins was recorded from m/z 350 to 6 50. Auto MS 3 was conducted with 100% compound stability and 100% trap drive level. An external standard mixture of 3 O glucosides of delphinidin, cyanidin, petunidin, pelargonidin, peonidin and malvidin was used to quantify anthocyanins. Pure compounds of Chlorogenic acid, rutin, myricetin, quercetin, and kaempferol were used as external standards to quantify chlorogenic acid and flavonols. Data was analyzed using Chemstation software (Version B. 01.03, Agilent Technologies, Palo Alto, CA). Blueberry ph ytochemicals were identified on the basis of full scan and product ion mass spectra, UV/vis spectra on diode array detector, and comparison with published papers (Cho et al., 2004) Statistical Analyses. Data were expressed as mean standard deviation. One way analyses of variance with Tukey Kramer HSD comparison of means were performed using JMP w as used to compare ABA treated samples w Results Berry Quality. Blueberries grow in small clusters of 9 12 berries. About 65 % and 75% of berries on cluste rs were ripe for Star variety at first and second sampling, respectively (Table 2 1). Ripening of blueberries is characterized by a significant size and weight increase firmness decrease, and development of blue/purple color. ABA at 400 ppm significantly decreased the percentage of ripe berries on Star variety at second sampling when

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30 compared to the control (p = 0.05). A significant decrease in the percentage of ripe berries was observed on Windsor variety treated with 200 ppm or 400 ppm ABA at first sampl ing, indicated a delay in ripening. The delay of ripening was also observed for Star at first sampling and Windsor at second sampling, however, the differences were not statistically significant. ABA significantly decreased the average weight of all berrie s on the cluster for Windsor compared to the control. There was also a significant decrease in the average weight of ripe berries for Windsor at second sampling, suggesting ABA may decrease berry yield. ABA significantly increased the firmness of all berri es for both effects on berry ripening. Ripe blueberries have a blue purple color and give a negative b* value using the L*, a*, and b* color scale. Unripe berries are yellow green and give a positive b* value. Table 2 1 shows that b* values increased for all berries on clusters in both Star and Windsor. A trend (p = 0.09) of delay in color development was observed only in the 400 ppm treated Star berries at the first s ampling. Nevertheless, ABA treated ripe berries had higher b* values than control ripe berries on Star variety. Similar effects were also observed on Windsor variety, but only during the second sampling. It appeared that in most of the berries, ABA made th e ripe blueberries less blue than the controls ( Table 2 1 ). Because the blue color of berries is caused by anthocyanins in the skin, this may have suggested a decrease of anthocyanin content. Total Phenolic Content and Antioxidant Capacity. There was no significant difference in the phenolic content or antioxidant capacity (measured by ORAC or DPPH) in control and ABA treated samples of both the varieties

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31 at the first sampling (Table 2 2). During second sampling, there was a significant decrease in antiox idant capacity measured by DPPH assay in 200 ppm and 400 ppm ABA treated samples for both varieties. Such decrease was consistent with a trend of decrease in total phenolic content in 400 ppm ABA treated Windsor variety (p=0.06). However, such trend was n ot observed in ORAC. No significant difference in total phenolic content or ORAC was shown for Star variety at second sampling. Phytochemical Identification and Q uantification. Twelve anthocyanins, a hydroxycinnamic acid, and five flavonols were identified and quantified in ripe berries of Star variety using HPLC ESI MS n ( Figure 2 1 ). Ripe berries of Windsor variety contained ten anthocyanins, a chlorogenic acid, and two flavonols. Malvidin 3 arabinoside, delphinidin 3 galactoside, and malvidin 3 galactoside were the major anthocyanins in Star variety. They contributed to 23%, 16%, and 15% of total anthocyanin content. Cyanidin 3 glucoside was the major anthocyanin in Windsor variety and accounted for 44% of total anthocyanins. Chlorogenic acid wa s the most abundant non anthocyanin phenolic compound in both varieties. ABA application did not affect the contents of anthocyanins, flavonols, or chlorogenic acid in both the varieties at each sampling time ( Tables 2 3 and 2 4 ). Discussion Two varieties of southern high bush blueberries were used for ABA applications. The berries were sampled twice i.e. after second and third ABA applications, respectively. The impact of ABA on berry quality appeared to be both sampling time and variety dependent. There was a clear effect of ABA to delay fruit ripening of southern high bush blueberries. A significant ripening delay was observed in Windsor variety

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32 during first sampling. This was reflected by a higher percentage of unripe berries and greater firmness of al l berries on the clusters. Blueberry is a non climacteric fruit whose ripening is regulated by abscisic a cid (Zhang, Leng, Zhang & Li, 2009) Endogenous ABA is associated with accelerated ripening because ABA increases rapidly at the beginning of fruit ripening and has a high correlation with the acc umulation of sugar, reduction of acid, and production of fruit pigments (Sun, Zhang, Ren, Qi, Zhang & Leng 2010) The ripening delay effects of exogenous ABA can be attributed to its function to inhibit plant growth. ABA inhibits photosynthetic CO 2 assimilation by inducing stomatal closure or the activity of ribulose 1,5 biophophate carboxylase (Seemann & Sharkey, 1987) The photosynthetic inhibitory effect of exogenous ABA may also explain the significant decrease in the weight of ripe berries for the Windsor variety. This obs ervation was consistent with a previous study which showed that exogenous ABA significantly decreased the yield of lettuce (Li et al., 2010) ABA consistently accelerated ripening of climacteric fruit, such as banana or tomatoes, by enhancing ethylene synthesis or the sensitivity of fruits to ethylene (Jiang, Joyce & Macnish, 2000; Zhang, Yuan & Leng, 2009) However, the effect of ABA in the ripening of non climacteric fruits appeared to be crop and variety specific. ABA did not impact fruit ripening on Muscadine grapes ( Vitis rotundifolia ), but it was reported that Vitis vinifera treated with ABA ripened faster than control (Giribaldi, Gny, Delrot & Schubert, 2010) different functions in different crops and at different growing stages of the same crops. Timing and dose of ABA appl ication are also important factors (Walton, 1980) There

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33 wa s a significant trend for ABA to increase in the firmness of all berries on clusters during the first sampling on both varieties. However, it did not affect the firmness of all berries during second sampling or the firmness of ripe berries. Increase of fir mness of all berries was consistent with a delay in ripening. These results agreed with a previous study which showed that texture of ABA treated blueberries was not significantly different from controls (Hussin & Basiouny, 1984) ABA did not affect total phenolic content of ripe berries. A similar observation was also made about the effects of ABA on phenolic content of low bush blueberries ABA had no impact on the antioxidant capacity of ripe blueberries measured using ORAC. However, ABA decreased the antioxidant capacity measured by DPPH for both varieties but only at the second sampling. Compared to the low bush blueberries, there was a considerable decrease in the number of anthocyanins in the southern high bush blueberries (Prior, Lazarus, Cao, Muccitelli & Hammerstone, 2001) This was consistent with a study in which low bush blueberries were shown to have 38% more anthocyanins and 54% higher ORAC values (Kalt, Ryan, Duy, Prior, Ehle nfeldt & Vander Kloet, 2001) The antioxidant capacity measured by DPPH was much lower than that measured by ORAC. This was because DPPH and ORAC methods used different chemistry and mechanisms to measure antioxidant capacity. ORAC assay applies a compe titive reaction scheme, in which antioxidant and substrates compete for thermally generated peroxyl radicals through the decomposition of azo compounds. DPPH assay measures the capacity of an antioxidant in the reduction of an oxidant, which changes color when reduced (Huang, O u & Prior, 2005) The lower values for DPPH could be attributed to

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34 the fact that those antioxidants that react quickly with peroxy radicals will react even slower or may not react with DPPH due to steric inaccessibility. ABA application did not affect anthocyanin, flavonol, and chlorogenic acid contents of ripe blueberries. These observations con tradicted other studies in which ABA significantly increased the anthocyanin content in red leaf lettuce and red grapes (Jiang & Joyce, 2003; Kondo, 1997; Li et al., 2010; Peppi, Fidelibus & Dokoozlian, 2006; Sandhu et al., 2011) These results however, agreed with those on lowbush blueberries where an ABA application showed no impact on anthocyanin content (Forney, Kalt, Abrams & Owen, 2009; Percival, 2007) ABA stimulated anthocyanin biosynthesis in crops because it induced the expression of several enzymes in the flavonoid bio synthetic pathway (Gagn, Cluzet, Mrillon & Gny, 2011) Fruit ripening is also accompanied by a rapid increase in the activity of these enzymes. This is evident for blueberries because the green berries turned dark blue and became ripe within three weeks. A major effect of ABA on blueberries appeared to be a ripening delay. This may have counteracted any capacity of ABA to increase enzyme expression for flavonoid biosynthesis. As a net effect, we saw no impact of ABA application on total phenolic content, antioxidant capacity, or flavonoid content of ripe berries. Conclusions ABA had a clear trend to delay the ripening of southern high bush blueberries and decreas ed berry yield in Windsor variety. ABA application did not affect total phenolic content, antioxidant capacity, or flavonoid content of ripe berries in Star variety. It showed a trend to decrease total phenolic content and antioxidant capacity of ripe berr ies in Windsor variety. The effects of ABA on berry quality were variety dependent and also varied according to sampling time

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35 Table 2 1 Fruit quality of blueberries as affected by ABA treatments Data are mean standard deviation for n=8. Means Kramer HSD pair

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36 Table 2 2 T otal phenolic content and antioxidant capacities of ripe blueberries as affected b y ABA treatment Data are mean standard deviation for n=8 for 1st sampling and n=5 for 2nd sampling (block 1 4 plus a pooled sample from block 5 8). For each variety, means within a column followed by the same letter are not significantly different at 0.05.

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37 Table 2 3 Content of anthocyanins, flavonols, and chlorogenic acid in ripe berries of Star variety as affected by ABA treatmen t Data are mean standard deviation for n=8 for 1st sampling and n=5 for 2nd sampling (block 1 4 plus a pooled sample from block 5 8). For each variety, means within a column followed by the same letter are not significantly different at 0.05. Results are represented as g/g fresh weight bas is.

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38 Table 2 4. Content of anthocyanins, flavonols, and chlorogenic acid in ripe berries of Windsor variety as affected by ABA treatments Data are mean standard deviation for n=8 for 1st sampling and n=5 for 2nd sampling (block 1 4 plus a pooled samp le from block 5 0.05. Results are represented as g/g fresh weight basis

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39 Figure 2 1 HPLC chromatograms phenolic compounds in blueberries of Star variety A) 360 nm detection. B) 520 nm detection Peaks were identified using mass spectrometry. (1) chlorogenic acid, (2) quercetin 3 galactoside, (3) quercetin 3 glucoside, (4) quercetin xyl oside, (5) myricetin 3 rhamnoside, (6) rutin, (7) delphinidin 3 galactoside (8) cyanidin 3 galactoside, (9) delphinidin 3 arabinoside, (10) petunidin 3 galactoside, (11) cyanidin 3 arabinoside, (12) peonidin 3 galactoside, (13) petunidin 3 arabinoside, ( 14) malvidin 3 galactoside, (15) ma lvidin 3 glucoside, (16) malvidin 3 arabinoside, (17) petunidin 3 acetyglucoside, (18) malvidin 3 acetylglucoside.

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40 CHAPTER 3 EXTRACTION AND CONCE NTRATION OF PHYTOCHE MICALS FROM B LUEBERRIES USING ULTRASOUND ASSISTED EXTRACTION AND RESIN ADSORPTION Introduction Fresh blueberries are known to have a higher antioxidant capacity than other fruits (Prior et al., 2007; Wu, Beecher, Holden, Haytowitz, Gebhardt & Prior, 2004) Phytochemicals from blueberries are shown to lower blood cholesterol (Prior et al., 2009) and prevent cancers and atherosclerosis (Adams, Phung, Yee, Seeram, Li & Chen, 2010) (Wu et al., 2010) Research suggested that phytochemicals extracted from blueberries were more effective than whole fruits in preventing body weight gain (Prior, Wu, Gu, Hager, Hager & Howard, 2008a) There is a consumer demand for concentrated blueberry phytochemicals in the form of a dietary supplement to boast antioxidant intake. Blueberries contain 1 0.8 g of sugar per 100 g of fresh berries. About 8.3% and 35% of the US population are diabetic or pre diabetic (CDC, 2011) This group of consumers can benefit from a concentrated phytochemical product from blueberries with little or no sugar content. Resin adsorption technology is being explored to concen trate polyphenols as well as for the removal of sugars (Kammerer, Gajdos Kljusuric, Carle & Schieber, 2005) Synthetic resins allow adsorption of polyphenols from aqueous solution via hydrophobic binding and aromatic stacking. They desorb phytochemicals in organic solvents, such as metha nol or ethanol. Because sugars do not interact with resins, they can be easily removed by water elution. Ultrasound was investigated to increase the extraction efficiencies. Polyphenols entrapped in the plant matrix were effectively released by ultrasound in a previous study (Chemat, Zill e & Khan, 2011) In the present study, the

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41 high extraction cap acity of ultra sound was coupled with the concentration capacity of resin adsorption to produce concentrated phytochemical extracts from blueberries. Materials and Methods Chemicals azotis(2 amidinopropane)) was a product of Wako Chemicals Inc. (Bellwood, RI). Trolox (6 hydroxy 2,5,7,8 tetramethylchroman 2 carboxylic acid), and cyanidin 3 rutinoside were purchased from Sigma Aldrich (St. Louis, MO). Folin Ciocalteu reagent, chlorogenic acid, and other chemicals were products of Fisher Scien tific (Pittsburg, PA). Amberlite resins (XAD 761, XAD 4, XAD 1180, XAD 7HP, and FPX 66) were products of Rohm Hass (Philadelphia, PA). Chemical and physical properties of these resins are summarized in Table 3 1. Pre Treatment of R esins Resins were suspen ded in DI Water to expand the beads before packed in a glass column (I.D. x L: 22 x 350 mm). Ethanol (140 mL) was used to wash the column with a flow rate of 400 mL/hr. Column was washed with water until the eluent was clear. The resin was then eluted with 140 mL of 4% HCl followed with distilled water until pH of the eluent was about 7.0. The column was the washed with 140 mL of 5% NaOH followed by distilled water until the eluent reached a pH of 7.0. After pre treatment, the resins were ready to be used. A sample of the pre treated resins were weighed into uncovered trays and placed in an oven and kept at 60 o C for 24 h r. Weight loss was calculated as moisture content. Extraction of Phytochemicals from B lueberries The extraction and concentration of blu eberry phytochemicals is depicted in figure 3 1. Frozen southern high bush blueberries (160 g) were mixed with 400 mL of hot

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42 water (40 o C, 60 o C, or 90 o C, acidified with 0.5% v/v acetic acid) and blended for 1 minute in a blender. An ultrasound probe was pla ced into the blueberry mash and mixture was sonicated at 100% amplitude for 0 or 5 min. The extraction mixture was filtered through cheese cloth to separate water extract and pomace. The pomace was re extracted with an additional 400 mL of acidified water by using ultrasound. The water extracts were combined and filtered through Whatmann No. 4 filter papers. The blueberry pomace after ultrasound assisted water extraction was extracted using a methanol/acetic acid (97.3:0.3, v/v). One gram of blueberry pom ace was combined with 10 mL of extraction solvent and vortexed for 30 sec. Samples were kept in darkness for 20 min and then centrifuged for 10 min at 20 o C and 3 200 rpm. The supernatant was removed and collected. For comparison, frozen blueberries were a lso extracted using organic solvents. Frozen blueberries (160 g) were blended with 400 mL of methanol/acetic acid (99.7:0.3, v/v) for 1 min. Samples were then vortexed for 30 sec and kept in the darkness for 20 min. Samples were then centrifuged for 10 min utes at 20 o C at 3 200 rpm. Ultrasound was not used for organic solvent extraction. Final extracts (WPB and MPB) were dried under vacuum conditions for 24 hr to ensure complete removal of organic solvents (Thermo Scientific Savant ISS110 Speed Vac. Concentrator; Waltham MA). Static Adsorption/Desorption T esting Pre treated hydrated res in (1 g ) and 25 mL of blueberry water extract were added to a 250 mL flask with stoppers and wrapped in aluminum foil. Flasks were placed in a water bath shaker (Stovall Life Sciences Inc. ; Greensboro, NC) at a rate of 45 rpm. Adsorption was continued at r oom temperature for 24 hr to ensure the adsorption

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43 equilibrium was reached For static desorption testing, th e phytochemical laden resin s were washed with 25 mL DI water in a flask After the rinsing water was discarded, 50 mL of 95% e thanol was added in t he flask. The flask was placed on a water bath shaker for 24 hr at room temperature at a rate of 45 rpm. Adsorption and desorption ratios and capacities were calculated using the following equations Adsorption Ratio: 3 1 Adsorption Capacity 3 2 Where A is the adsorption ratio (%) and q e is the adsorption capacity (mg/g dry resin) at equilibrium. C o and C e are the initial and equilibrium concentrations of the blueberry extract solutions (mg/L). M is the moisture content of the resin (%) where as W is the initial weight of the resin being used (g). V i is the volume (mL) of blueberry ex tract used Desorption Ratio 3 3 Desorption Capacity 3 4 % Recovery 3 5 Where D is the desorption ratio (%), q d is the desorption capacity (mg/g dry resin), and R is the % Recovery (%) after desorption is complete. C d is the concentration of

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44 blueberry in the desorption solution (mg/L). V d is the volume of the desorption solution (mL). Co, C e M, W, and V i are the same as a bove. Adsorption K inetics Pre treated resin ( 1 g ) was mixed with 25 mL of blueberry water extract in a flask and placed on a water bath shaker An aliquot of s upernatant (1 ml) was obtained every 30 min for the first 6 hr and then every 60 min from 6 to 12 hr A dsorption kinetic s were evaluated using the pseudo first and second order models. Pseudo First Order Model 3 6 Pseudo Second Order Model 3 7 Where k f i s the rate constant of the pseudo first order m odel and ks is the rate constant of the pseudo second order m odel. Where q t (mg/g) is the amount of adsorbate adsorbed at time t where q e is the adsorption capacity at equilibrium. Adsorption Isotherms and T hermodynamics FPX 66 r esin was selected for adsorption Isotherms and thermodynamics testing. Hydrated resin (1 g) was added to blueberry water extracts of different concentrations. Temperature was set at 25, 35, or 45 o C. The equilibrium adsorption isotherms for total phenolic s and total anthocyanins were determined using Langmuir and Freundlich equations. Langmuir Equation 3 8

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45 Where K L L are equilibrium constants of the Langmuir Equation in which plotting C e /q e results in a straight line having the slope of q/K L and an intercept of 1/ K L. Freundlich Isotherm 3 9 Where by potting log q e vs log C e and a constant K F and an exponent of 1/n can be calculated Dynamic Adsorption/Desorption T esting A glass column with a fritted disk (I.D. x L, 19 x 400 mm) was loaded with FPX 66 resin with a resin bed volume of 30 mL. Blueberry water extract was loaded into this column using a flow rate of 2, 5 or 10 BV/hr, respectively The adsorbate laden column was then washed with DI water (150 mL) to remove sugars and other compounds that did not absorb on the resin. Phytochemicals were desorbed using 95% ethanol at a flow rate 2, 4, or 6BV/hr, respectively. Eluent was collected at each 30 mL and analyzed for total anthocyanins. Folin Ciocalteu A ssay Total phenolic content was determined by the Folin Ciocalteu assay. Blueberry extracts were mixed with diluted Folin Ciocalteu reagent and 15% sodium carbonate. Absorbance at 765 nm was measured on a SPECTRAmax 190 microplate reader (Molecular Devices, Sunnyvale, CA) after 30 min of incubation at room temperature. Gallic acid was used to generate a standard curve. Results of total phenolic content for blueberries were expressed as millig ram gallic acid equivalent per gram of fresh blueberry samples (mg GAE/g).

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46 Total A nthocyanin C ontent Total anthocyanin content was measured by using a pH differential assay ( Giusti & Wrolstad, 2001 ) Absorbance at 520 nm and 700 nm were measured on a Life Science UV/Vis spectrophotometer (DU 730, Beckman Coulter, Fullerton, CA) after 15 min of incubation at room temperature. Absorbance (A) was calculated using (A520 A700) pH 1.0 (A520 A700) pH 4.5. Total anthocyanin content (mg Cy G/g) was calculated using (A 449.0 80 1000) / (2974 0 1) for blueberry and were expressed as milligram cyanidin 3 glucoside equivalent per gram of fresh blueberry (mg Cy G/g). Total Procyanidin C ontent Total procyanidin content was determined using 4 dimethylaminocinnamaldehyde (DMAC) colorimetric method (Prior et al., 2010) An aliquot of 70 l of blueberry extract was mixed with DMAC solution (0.1%, 210 l) in a 96 well plate. Epicatechin series dilutions with concentrations ranging from 0 50g/ml were used to generate a standard curve. Absorption at 640 nm was measured on a microplate reader (SPECTRAmax 190, Molecular Devices, Sunnyvale, CA) after 30 min of incubation in darkness. Results of total procyanidins were expressed as micrograms epicatechin equivalents per milligram of sugar free extract (g epicatechin/g). Oxygen Radical Absorbance C apacity (ORAC FL ) A ssay Blueberry extracts were incubated with fluorescein as a free radical prob e and AAPH as a free radical generator (Prior et al., 2003) The kinetics of fluorescein degradation was read on a Spectra XMS Gemini microplate reader (Molecular Devices, Sunnyvale, CA). 6 Hydroxy 2,5,7,8 tetramethylchroman 2 carboxylic acid (Trolox) was used to generate a standard curve. The results of the antioxidant capacity of blueberries

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47 were expressed as mol Trolox equivalent per gram of fresh blueberry samples (mol TE/g). HPLC Analyses of Phytochemicals and S ugars An Agilent 1200 HPLC system consisting of an autosampler, a binary pump, a column compartment, a diode array detector and a refractive index detector (Agilent Technologies, Palo Alto, CA) was interfaced to a HCT ion trap mass spectrometer (Bruker Daltonics, Billerica, MA). Samples were centrifuged at 13300 rpm for 10 min and 10L supernatant was injected for phytochemical analysis. A Zorbax SB C18 ogies, Palo Alto, CA) was used for the separation. The binary mobile phase consisted of (A) formic acid: water (5:95 v/v) and (B) methanol. For the analysis of flavonols, chlorogenic acid derivatives and anthocyanins, a 70 min gradient was adapted from a p ublished paper (Cho et al., 2004) The gradient is described as follows: 0 min 5% B, 2 min 5% B, 10 min 20% B, 15 min 20% B, 30 min 25% B, 35 min 25% B, 50 min 33% B, 55 min 40% B, 60 min 60% B, 65 min 70% B, 70 min 5% B isocratic; followed by 5 min of re equilibration of the column before the next run. The detection wavelength was 520 nm for anthocyanins, and 360 nm for chlorogeni c acid and flavonols. Electrospray ionization in alternating mode was performed using nebulizer 30 psi, drying gas 11 L/min, drying temperature 300 o C, and capillary of 4000 V allowing compounds to be detected in both positive and negative mode in the same run. The full scan mass spectra of the anthocyanins was recorded from m/z 350 to 650. Auto MS3 was conducted with 100% compound stability and 100% trap drive level. Pure compounds of c hlorogenic acid, rutin, myricetin, quercetin, and kaempferol were used a s external standards to quantify chlorogenic acid and flavonols. Cyanidin 3 rutinoside was used as an external standard

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48 for the quantification of anthocyanins. Data was analyzed using Chemstation software (Version B. 01.03, Agilent Technologies, Palo Alto, CA). Blueberry phytochemicals were identified on the basis of full scan and product ion mass spectra, UV/VIS spectra on diode array detector, and comparison with published papers (Wu & Prior, 2005) (Cho et al., 2004) Sugar analysis was conducted using a Resteck ultra amino column (5 m, 250 x 4.6mm). Acetonitrile: water (65:35 v/v) was used as the mobile phase at a constant flow rate of 1.0 mL/min. The column temperature was maintained at 30 o C and 5 L of sample was in jected. Calibration curves were constructed using pure standards of glucose and fructose. Statistical Analyses Data were expressed as mean standard deviation. One way analyses of variance with Tukey Kramer HSD comparison of means were performed using J MP considered as significant. Results and Discussion Extraction of Polyphenols Using Power U ltrasound Blueberries contain ant h ocyanins, procyanidins, and flavonols. Antho cyanins are water soluble, whereas procyanidins and flavonols are partially water soluble. Aqueous alcohol or acetone is commonly used to extract polyphenols. However, the use of organic solvents faces safety and environmental issues. We sought to use wate r as an extraction solvent and improve its extraction efficiency with higher temperature and ultrasound.

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49 Water alone at 40 o C, 60 o C, 90 o C extracted similar amounts of total phenolics and total procyanidins from frozen fresh blueberries (Table 3 2). Water a t 90 o C extracted more anthocyanins than did water at 40 o C or 60 o C. Sonication drastically enhanced the extraction efficiently of polyphenols by wa ter at all three temperatures. The amount of total procyanidins or total anthocyanins extracted by 90 o C water was double d by sonication. Sonication assisted hot water extraction at 90 o C showed the highest extraction efficienc than that of methanol. The total anthocyanin content of blueberri es in the present study was 1.45 mg/g using methanol extraction and a pH differential method. This concentration was similar to those rep orted in a published study (1.28 1.87 mg/g) (Rodriguez Mateos, Cifuentes Gomez, Tabatabaee, Lecras & Spencer, 2011) Total phenolic content ( 2.22 mg/g) was comparable with pre viously reported values (2.61 5.33 mg/g ) in high bu sh blueberries (Sellappan et al., 2002) Power ultrasound was known to increase extraction ef ficiency by form of a cavitation bubble in the extraction medium. These bubbles are generated on the plant at the material surface. A compression cycle during sonication causes the bubble to collapse that creates a micro jet toward the plant surface. It ru ptures the cell wall of the plant and releases cell content into the medium (Chemat et al., 2011) Static Adsorption and D esorption Adsorption and desorption behavior of anth ocyanins in resins are depicted in Figure 3 2. Amberlite resin FPX 66 and XAD 1180 had an adsorption ratio at 99.8% and 96.7%, respectively (Figure 3 2 A). XAD 761 showed the lo west at 79.8%. XAD 7HP was determined to have the highest adsorption capacity (25.8 mg/g) with FPX 66 and

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50 XAD 1180 following at 24.1 and 23.9 mg/g, respectively. XAD 4 had the lowest adsorption capacity at 17.9 mg/g when compared to the other resins that w as not expected as XAD 4 had the highest surface area out of all the resins at 700 m 2 /g. that may explain its low adsorption capacity. Previous research suggested that the surface area in combin ation with pore size were main determining factors in predicting the adsorption capacity (Xu, Zhang, Chen, Wang & Ande rson, 1999) In desorption tests, FPX 66 and XAD 761 had a higher desorption ratio (121.1% and 119.9%) and greater recovery than the other three resins. Both FPX 66 and XAD 761 have large particle sizes at 0.75 mm and 0.70 mm, respectively. Resins with larger particl e sizes tend to have higher mass exchange rates allowing more material to be transferred to and from the resin, therefore increasing the desorption ratio and recovery (Bathen & Breitbach, 2001; Worch, 1990) Figure 3 3 describes the adsorption and desorption behaviors of total phenolic content. XAD 761 had the highest adsorption ratio as well as the highest adsorption capacity at 88.6% and 53.4 mg/g resp ectively. XAD 7HP had the lowest adsorption ratio at 70.6%, whereas FPX 66 and XAD 4 were between. In contrast to a high adsorption ratio, XAD 761 had the lowest desorption ratio of 21.0% and a recovery of only 16.5%. This suggested that significant amount of polyphenols were adsorbed irreversibly on XAD 761. FPX 66 was the most efficient resin in desorption with recovery of 82.5% and a desorption ratio of 114.9%. Figure 3 4 shows adsorption and desorption behaviors of total procyanidins. FPX 66, XAD 1180 and XAD 4 showed the highest adsorption capacity and adsorption ratio. XAD 1180 had the lowest recovery and desorption ratio out of all the resins. FPX 66

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51 and XAD 7HP had a recovery of 80.4% and 75.6% and a desorption ratio of 92.4% and 89.8% respectivel y. FPX66, XAD 7HP and XAD 4 were all consistently more efficient at adsorption and desorption than other two resins, hence they were chosen for further kinetic tests. Adsorption k inetics Figure 3 5A shows the adsorption kinetics of anthocyanins on three r esins. FPX 66 and XAD 7HP reached equilibrium at a pproximately 6 hr, which was faster than XAD 4 (about 8 hours). At equilibrium, FPX 66 had higher adsorption capacity than XAD 7HP or XAD 4. Regression of kinetic data using pseudo first order model render ed straight lines between log (q e q t ) and time (Figure 3 5B). The correlation coefficients lie between 0.954 0.988 (Table 3 3). Regression of kinetic data using pseudo second order models resulted straight lines between t/q t and time. The correlation coeff icients were 0.987 0.997 (Table 3 3). The pseudo first order model describes the initial stages of the adsorption process. The pseudo second order model is more useful to predict and describe the entire adsorption process. The pseudo second order model app eared to be better than pseudo first order in describing the adsorption kinetics of anthocyanins on resins. Similar observations were made in previous studies using Amberlite resins (Abdullah, Chiang & Nadeem, 2009) Figure 3 6A illustrates the adsorption kinetics of total phenolics on resins. Ad sorption capacity of FPX 66 and XAD 7HP increased sharply within the first 3 hr, whereas a gradual increase was seen on XAD 4. At 6 hr both the FPX 66 and XAD 7HP resins reached equilibrium with FPX 66 having a higher adsorption capacity. At the end of 720 min XAD 4 matched the adsorption capacity of FPX 66. Regression of kinetic data using pseudo first and second order models is depicted in Figure 3 6B and 3 6C.

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52 The adsorption kinetics of procyanidins appeared to be different than anthocyanins (Figure 3 7 A). FPX 66 had the lowest adsorption capacity when compared to the other two resins. XAD 7HP showed the highest adsorption capacity for procyanidins. This may be explained by the fact that procyanidin compounds are much larger than anthocyanins and need a larger pore diameter for adsorption. With surface area and pore diameter being inversely proportional to one another, we can conclude that XAD 7HP having the smaller surface area of the three resins tested would in turn have a larger pore diameter and ther efore, be more susceptible to the diffusion of the larger procyanidin compounds than the other two resins ( Worch, 1990 ) Regression of kinetic data using pseudo first and second order models is depicted in Figure 3 7B and 3 7C. Rate constants and correlation coefficients based on total phenolics, total anthocyanins and total procyanidins are shown in Table 3 3. Adsorption i sotherms and thermody namics of FPX 66 r esin Figure 3 8A shows the adsorption isotherms of anthocyanin on FPX 66 at 25 o C, 35 o C, and 45 o C). Data was regressed according to the Langmuir (Figure 3 8B) and Freundlich isotherms equations (Figure 3 8C). Equation constants and corre lation coefficients are listed in Table 3 4. The Langmuir and Freundlich isotherms are the most common models for exploring adsorption equilibrium data. The Freundlich model assumes that the surface of the resin is heterogeneous in nature that is character ized by sorption sites at different energies. This describes the adsorption behavior of a monomolecular layer as well as a multi molecular layer. The Langmuir model describes a monolayer adsorption with energetically identical sorption sites as well as wit hout mutual interactions between the adsorbed molecules (Duran, Ozdes, Gundogdu & Senturk, 2011) The correlation coefficients in Table 3 4 showed that the Langmuir

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53 Model was a better fit for the data, suggesting that adsorption of anthocyanins was endothermic in nature ( Gkmen & Serpen, 2002 ) Results based on total anthocyanins show the maximum adsorption capacity (q e ) remains consistent implying that temperature did not affect the adsorption capacity. However, the K L values decrease as temperature increases suggesting adsorption of anthocyanins was decreased as temperature rose. Degradation of the heat sensitive anthocyanins at higher temperatures may explain these results. Adsorption isotherm of total phenolics and are shown in Figure 3 9. In contrast to the isotherm data on anthocyanins, both the Langmuir equation and Freundlich equation fitted the data very well (Figure 3 9 B,C and Table 3 4). The m aximum adsorption capacity (q e ) of total phenolics as well as the K L values were not affected by an increase in temperature. For both anthocyanins and total phenolics, FPX 66 Resin had the highest adsorption efficiency at lowest temperature (25 o C) ( W. L McCabe J.C. Smith, 1993 ) Dynamic A dsorption/ D esorption on FPX 66 Resin When blueberry water extract was loaded into a FPX 66 resin column, no anthocyanins could be detected in the eluent at the beginning (Figure 3 10). After more extract was loaded, the resins in column slowly reached adsorption saturation. Anthocyan ins started to breakthrough and would appear in the eluent. Breakthrough volume is defined as the volume of extract loaded on the column when the concentration of anthocyanins in the eluents is 5% of that in water extract At a flow rate of 10 BV /hr and 5 BV/hr, the breakthrough volumes were 8 BV and 16 BV respectively. At a flow rate of 2 BV /hr, breakthrough was not reached after 23 BV of extract was loaded on the column. The adsorption ratios of anthocyanins were 98.2%, 89.3%, or

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54 82.2% respectively usi ng a flow rate of 2BV/hr, 5BV/hr, or 10 BV/hr Similar results were reported in an earlier study that investigated the adsorption of lycopene on resin s (Liu, Liu, Chen, Liu & Di, 2010) The contact time between anthocyanins and resin became shorter when the flow rate increase d Shorter contact time subs equently led to a less complete adsorption (Ma et al., 2009) Results suggested that 5 BV/h r was a suitable flow rate as it allowed for maximum adsorption of phenolic compounds in the shortest amount of ti me. E thanol (95%, v/v) was used to desorp polyphenols from column using a flow rate of 2, 4, or 6 BV/h (Figure 3 11) Regardless of flow rate, a complete desorption of anthocyanins were achieved with less than 3 BV of ethanol. In this case, one should c hoose the highest flow rate (6 BV/hr), because this would result in the shortest time used for desorption. Phytochemical and Sugar Composition in E xtracts H ot water extract of blueberries contain ed fructose and glucose. Sugar was not detected in WBP or M BP using HPLC analysis. Concentration of anthocyanins, flavonols and chlorogenic acid are shown in Table 3 5 Individual anthocyanin content in WBP was higher than in MBP and fresh blueberries. The WBP contain ed 100 times more chlor o genic acid than in the fresh berries and MBP. WBP also contained more flavanols than both the control and MBP extract. Yield and R ecovery of P olyphenols WBP and MBP had a yield of 1.22 g/100 g berries or 1.39 g/100 g berries, respectively (Table 3 6). The ORAC of the WBP was about 100 fold of fresh blueberries and about 300 fold of that of the MBP. The total phenolic content in WBP was about 100 fold of that in fresh blueberries. Interestingly, total phenolic content in fresh berries was

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55 higher than that of MBP, suggesting majority of phenolic compounds were extracted into WBP. This was consistent with the extraction data. The total anthocyanins followed similar trend to those of total phenolic compounds. Tot al procyanidin contents were concentrated by 10 0 fold in WBP when compared to fresh berries. The recovery rates of total phenolics were 69.5 % in WBP and 0.52 % in MBP. About 90 % of total anthocyanins were recovered in WBP. Conclusion Application of ultraso und assisted extraction and resin adsorption yield two concentrated phytochemical extracts from fresh blueberries Antioxidant capacities, total phenolic, anthocyanin contents in the WBP extract were much higher than in the fresh blueberries. The MBP yiel ded lower values when compared to fresh berries suggesting the majority of compounds were extracted into the WBP. Sugars were not detected in either of these extracts ; t h us products are suitable as a dietary supplement for over 40% of the US with diabetes or pre diabetes.

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56 Table 3 1 Chemical and physical properties of resins

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57 Table 3 2 Extraction efficiency of different solvents under different extraction conditions Results are mean standard deviation of two determinations on fresh weight basis. Different superscripts in each column indicate the significant differences in the mean at

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58 Table 3 3. Pseudo first and second order rate constants of resins calculated on the basis of total phenolics, to tal anthocyanins and total procy anidins

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59 Table 3 4. Langmuir and Freundlich equation constants on Amberlite FPX 66 resin

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60 Table 3 5. Content of individual phenolic compounds in fresh blueberries, WBP, and MBP Data are mean standard deviation for three determinations. For each extract, means within a column followed by the same letter are not signific = Below Detectable Limits.

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61 Table 3 6. Yield, ORAC, total phenolic content in fresh blueberries, WBP and MBP Fresh blueberries WBP MBP Yield (g/100 g Fresh Blueberries) 1.22 0.01 1.39 0.76 ORAC Values (mole Trolox eq/g ) 68.3 0.71 7660 1.09 22.4 0.22 Total Phenolic Content (mg GAE/g extract) 3.77 .120 215 2.13 1.42 0.01 Recovery rate of total Phenolic, % 69.5% 0.52% Total Procyanidin Content ( g epicatechin/g extract) 0.108 5x10 3 2.37 0.02 0.08 2x10 3 Recovery of total procyanidin, % 26.7% 1.03% Total Anthocyanin Content ( mg Cy G/g extract) 1.86 0.05 139 1.01 0.92 0.03 Recovery of total anthocyanins, % 91.5% 0.69% Data are mean standard deviation for four determinations

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62 Figure 3 1. Flow chart for the extraction and concentration of blueberry phytochemicals.

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63 Figure 3 2. Static adsorption and desorption results based on total a nthocyanins. A) static adsorption capacity a nd rate of total anthocyanins. B) static desorption capacity and rate of total anthocyanins. Results are mean of three determinations. Different upper case letters indicate significant differences of bars (p < 0.05). Different lower case letters indicate significant differences of lines (p < 0.05). A B

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64 Figure 3 3. s tatic a dsorption and d esorption r esults b ased on t otal p henolics A) static adsorption capac ity and rate of total phenolics. B) static desorption capacity and rate of total phenolics. Results are mean of three determinations. Different upper case letters indicate significant differences of bars (p < 0.05). Different lower case letters indicate significant differences of lines (p < 0.05) A B

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65 Figure 3 4. S tatic a dsorption and d esorption r esults b ased on t otal procyanidins A) static adsorption capacity and rate of total procyanidins. B) static desorption capacity and rate of total procyanidins. Results are mean of three determinations. Different upper case letters indicate significant differences of bars (p < 0.05). Different lower case letters indicate significant differences of lines (p < 0.05). A B

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66 Figure 3 5. Kinetic curves of total anthocyanins on Amberlite resin FPX 66, XAD 4, and XAD 7HP A) adsorption k inetic curve B) pseudo first order kinetic curve C) pseudo second order kinetic curve A B C

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67 Figure 3 6. Kinetic curves of total phenolics on A mberlite resin FPX 66, XAD 4, and XAD 7HP A) adsorption k inetic curve. B) pseudo first order kinetic curve. C) pseudo second order kinetic curve. A B C

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68 Figure 3 7 Kinetic c urves of total procyanidins on A mberlite resin FPX 66, XAD 4, and XAD 7HP A) A dsorption k inetic curve s. B) Pseudo first order kinetic curve. C) P seudo second order kinetic curve. A B C

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69 Figure 3 8. Adsorption i sotherms and thermodynamics of total anthocyanins on Amber l ite resin FPX 66 A) adsorption isotherms curve. B) L angmuir isotherm regression curve. C) Freundlich isotherm regression curve A B C

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70 Figure 3 9. Adsorption i sotherms and thermodynamics of total phenolics on Amberlite resin FPX 66 A) Adsorption isotherms curve. B) langmuir isotherm regression. C) Freundlich isotherm regressio n A B C

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71 Figure 3 10. Dynamic adsorption curves of total anthocyanins on Amberlite resin FPX 66 at different flow rates Figure 3 11. Dynamic deso rp tion curves of total anthocyanins on Amberlite resin FPX 66 at different flow rates

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72 Figure 3 12. HPLC chromatogram of sugars in hot water extract using ref l ective index detection. Peak 1 and 2 were fructose and glucose, respectively. Figure 3 13. HPLC chromatogram of anthocyanins in sugar free resin produced blueberry extract at 520 nm detection Peaks were identified using mass spectrometry. 1) peonidin 3 glucoside, (2) cyanidin 3 galactoside, (3) peonidin 3 arabinoside, (4) petunidin 3 galactoside, (5) cyanidin 3 arabinoside,(6) peonidin 3 galactoside, (7) petunidin 3 arabinoside, ( 8) malvidin 3 galactoside, (9) malvidin 3 glucoside, (10) malvidin 3 arabinoside

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73 CHAPTER4 FINAL CONCLUSIONS ABA had a clear trend to delay the ripening of southern high bush blueberries and decreased berry yield in Windsor variety. ABA application did not affect total phenolic content, antioxidant capacity, or flavonoid content of ripe berries in Star variety. It showed a trend to decrease total phenolic content and antioxidant capacity of ripe berries in the Windsor variety. The effects of ABA on berry quality were variety dependent and also varied according to sampling time Application of ultrasound assisted extraction and resin adsorption yield two concentrated phytochemical extracts from fresh blueberries. Antioxidant capacities, total phenolic, ant hocyanin contents in the WBP extract were much higher than in the fresh blueberries. The MBP yielded lower values when compared to fresh berries suggesting the majority of compounds were extracted into the WBP. Sugars were not detected in either of these e xtracts allowing t he products to be suitable as a dietary supplement for those with diabetes or pre diabetes. Future development in this area could turn a low value, late season blueberry into a high margin blueberry extract. This product would not only b e applicable to those with diabetes but could also be marketed to the general public. Next steps would be to scale up to a pilot size production level and develop a cost effective product development process. Final product evaluation would also be necessar y to fully understand the impact the extract has on the human body. Sensory testing as well as glycation testing would be beneficial to understand the full capacities of this newly developed blueberry extract.

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74 LIST OF REFERENCES Abdullah, M. A., Chiang, L., & Nadeem, M. (2009). Comparative evaluation of adsorption kinetics and isotherms of a natural product removal by Amberlite polymeric adsorbents. Chemical Engineering Journal, 146(3), 370 376. Adams, L. S., Phu ng, S., Yee, N., Seeram, N. P., Li, L., & Chen, S. (2010). Blueberry phytochemicals inhibit growth and metastatic potential of MDA MB 231 breast cancer cells through modulation of the phosphatidylinositol 3 kinase pathway. Cancer Res, 70(9), 3594 3605. And ersen, M., & Markham, K. R. (2006). Flavonoids; Chemistry, Biochemistry and Applications. Boca Raton: CRC Press. Bailey, C. J. (1999). Insulin resistance and antidiabetic drugs. Biochemical Pharmacology, 58(10), 1511 1520. Basu, A., Rhone, M., & Lyons, T. J. (2010). Berries: emerging impact on cardiovascular health. Nutrition Reviews, 68(3), 168 177. Bathen, D., & Breitbach, M. (2001). Adsorptionstechnik,. Berlin. Baumann, K. (2010). Signalling: ABA's greatest hits. Nat Rev Mol Cell Biol, 11(1), 2 2. Bay nes, J. W., & Thorpe, S. R. (1999). Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes, 48(1), 1 9. Boyle, W. A., & Leone, M. (2008). Vasopressin in Septic Shock. New England Journal of Medicine, 358(25), 273 6 2738. Brand Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. LWT Food Science and Technology, 28(1), 25 30. Camire, M. E. (2000). Bilberries and blueberries as functional foods and nutra ceuticals in: Herbs, Botanicals & Teas, Technomic. Lancaster, PA: Technomic Publishing Company pp. 289 319. CDC (2011). National diabetes fact sheet: national estimates and general information on diabetes and prediabetes in the United States, 2011. Atlanta GA: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Chemat, F., Zill e, H., & Khan, M. K. (2011). Applications of ultrasound in food technology: Processing, preservation and extraction. Ultrasonics Sonochemistry, 18(4), 813 835. Cho, M. J., Howard, L. R., Prior, R. L., & Clark, J. R. (2004). Flavonoid glycosides and antioxidant capacity of various blackberry, blueberry and red grape genotypes determined by high performance liquid chromatography/mass spectrometry. Journal of the Science of Food and Agriculture, 84(13), 1771 1782.

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75 Duran, C., Ozdes, D., Gundogdu, A., & Senturk, H. B. (2011). Kinetics and Isotherm Analysis of Basic Dyes Adsorption onto Almond Shell (Prunus dulcis) as a Low Cost Adsorbent. Journal of Ch emical & Engineering Data, 56(5), 2136 2147. Forney, C. F., Kalt, W., Abrams, S. R., & Owen, S. J. (2009). Effects of postharvest light and ABA treatments on the composition of late harvested white cranberry fruit. ISHS Acta Horticulturae 810: IX Internati onal Vaccinium Symposium, 810, 799 806. Freeman, B. A., & Crapo, J. D. (1982). Biology of disease: free radicals and tissue injury. Lab Invest, 47(5), 412 426. Gagn, S., Cluzet, S., Mrillon, J. M., & Gny, L. (2011). ABA initiates anthocyanin production in grape cell cultures. Journal of Plant Growth Regulation, 30(1), 1 10. Giribaldi, M., Gny, L., Delrot, S., & Schubert, A. (2010). Proteomic analysis of the effects of ABA treatments on ripening Vitis vinifera berries. Journal of Experimental Botany, 61( 9), 2447 2458. Giusti, M. M., & Wrolstad, R. E. (2001). Characterization and Measurement of Anthocyanins by UV Visible Spectroscopy. John Wiley & Sons, Inc. Gkmen, V., & Serpen, A. (2002). Equilibrium and kinetic studies on the adsorption of dark colored compounds from apple juice using adsorbent resin. Journal of Food Engineering, 53(3), 221 227. Grace, M. H., Ribnicky, D. M., Kuhn, P., Poulev, A., Logendra, S., Yousef, G. G., Raskin, I., & Lila, M. A. (2009). Hypoglycemic activity of a novel anthocyanin rich formulation from lowbush blueberry, Vaccinium angustifolium Aiton. Phytomedicine, 16(5), 406 415. Harborne, J. B., & Williams, C. A. (2000). Advances in flavonoid research since 1992. Phytochemistry, 55(6), 481 504. Head, K. A. (2001). Natural therapi es for ocular disorders, part two: cataracts and glaucoma. Altern Med Rev, 6(2), 141 166. Hertog M., K. D. A. C., & et al. (1995). FLavonoid intake and long term risk of coronary heart disease and cancer in the seven countries study. Archives of Internal M edicine, 155(4), 381 386. Hooper, L., & Cassidy, A. (2006). A review of the health care potential of bioactive compounds. Journal of the Science of Food and Agriculture, 86(12), 1805 1813. Hou, D. X., Yanagita, T., Uto, T., Masuzaki, S., & Fujii, M. (2005) Anthocyanidins inhibit cyclooxygenase 2 expression in LPS evoked macrophages: Structure activity relationship and molecular mechanisms involved. Biochemical Pharmacology, 70(3), 417 425.

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76 Huang, D., Ou, B., & Prior, R. L. (2005). The Chemistry behind Anti oxidant Capacity Assays. Journal of Agricultural and Food Chemistry, 53(6), 1841 1856. Hussin, M. M., & Basiouny, F. M. (1984). The use of metabolic inhibitors, film forming antitranspirants, and Max I Jet Irrigation to increase yield, improve quality and water use efficiency of blueberries. Proceedings of the Florida State Horticultural Society(97), 348 350. IOM (2001). Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon Vanadium, and Zinc. The National Academies Press. Jiang, Y., & Joyce, D. C. (2003). ABA effects on ethylene production, PAL activity, anthocyanin and phenolic contents of strawberry fruit. Plant Growth Regulation, 39(2), 171 174. Jiang, Y., Joyce, D. C., & Macnish, A. J. (2000). Effect of abscisic acid on banana fruit ripening in relation to the role of ethylene. Journal of Plant Growth Regulation, 19(1), 106 111. Kalt, W., Ryan, D. A. J., Duy, J. C., Prior, R. L., Ehlenfeldt, M. K., & Vander Kloet, S. P. (2001). Interspecific Variation in Anthocyanins, Phenolics, and Antioxidant Capacity among Genotypes of Highbush and Lowbush Blueberries (Vaccinium Section cyanococcus spp.). Journal of Agricultural and Food Chemistry, 49(10), 4761 4767. Kammerer, D., Gaj dos Kljusuric, J., Carle, R., & Schieber, A. (2005). Recovery of anthocyanins from grape pomace extracts (<i>Vitis vinifera</i> L. cv. Cabernet Mitos) using a polymeric adsorber resin. European Food Research and Technology, 220(3), 431 437. Kon do, S. I., K. (1997). Abscisic acid (ABA) and 1 aminocyclopropane 1 carboxylic acid (ACC) content during growth of Satohnishiki cherry fruit, and the effect of ABA and ethephon application on fruit quality. Journal of Horticultural Science, 72(2), 221 227. Li, Z., Zhao, X., Sandhu, A. K., & Gu, L. (2010). Effects of exogenous abscisic acid on yield, antioxidant capacities, and phytochemical contents of greenhouse grown lettuces. Journal of Agricultural and Food Chemistry, 58(10), 6503 6509. Liu, Y., Liu, J. Chen, X., Liu, Y., & Di, D. (2010). Preparative separation and purification of lycopene from tomato skins extracts by macroporous adsorption resins. Food Chemistry, 123(4), 1027 1034. Luzuriaga, D. A., Balaban, M. O., & Yeralan, S. (1997). Analysis of vi sual quality attributes of white shrimp by machine vision. Journal of Food Science, 62(1), 113 118. Ma, C., Tao, G., JianTang, Lou, Z., Wang, H., Gu, X., Hu, L., & Yin, M. (2009). Preparative separation and purification of rosavin in Rhodiola rosea by macr oporous adsorption resins. Separation and Purification Technology, 69(1), 22 28. Macheix, J. J., Fleuriet, A., & Billot, J. (1990). Fruit Phenolics. CRC Press.

PAGE 77

77 Manach, C., Mazur, A., & Scalbert, A. (2005). Polyphenols and prevention of cardiovascular disea ses. Current Opinion in Lipidology, 16(1), 77 84. Martineau, L. C., Couture, A., Spoor, D., Benhaddou Andaloussi, A., Harris, C., Meddah, B., Leduc, C., Burt, A., Vuong, T., Mai Le, P., Prentki, M., Bennett, S. A., Arnason, J. T., & Haddad, P. S. (2006a). Anti diabetic properties of the Canadian lowbush blueberry Vaccinium angustifolium Ait. Phytomedicine, 13(9 10), 612 623. Martineau, L. C., Couture, A., Spoor, D., Benhaddou Andaloussi, A., Harris, C., Meddah, B., Leduc, C., Burt, A., Vuong, T., Mai Le, P. Prentki, M., Bennett, S. A., Arnason, J. T., & Haddad, P. S. (2006b). Anti diabetic properties of the Canadian lowbush blueberry Vaccinium angustifolium Ait. Phytomedicine, 13(9 10), 612 623. Norlin, S., Ahlgren, U., & Edlund, H. (2005). Nuclear factor Cells is required for glucose stimulated insulin secretion. Diabetes, 54(1), 125 132. Peppi, C. M., Fidelibus, M. W., & Dokoozlian, N. (2006). Abscisic acid application timing and concentration affect firmness, pigmentation, and color of 'F lame Seedless' grapes. American Society for Horticultural Science, 43(1), 173 176. Percival, D. M., Joanna L. (2007). Use of plant growth regulator to increase polyphenolic compounds in the wild blueberry. Agricultural Institute of Canada, 87(2), 333 336. Pickup, J. C. (2004). Inflammation and activated innate immunity in the pathogenesis of Type 2 Diabetes. Diabetes Care, 27(3), 813 823. Prior, R. L., Fan, E., Ji, H., Howell, A., Nio, C., Payne, M. J., & Reed, J. (2010). Multi laboratory validation of a st andard method for quantifying proanthocyanidins in cranberry powders. Journal of the Science of Food and Agriculture, 90(9), 1473 1478. Prior, R. L., Gu, L., Wu, X., Jacob, R. A., Sotoudeh, G., Kader, A. A., & Cook, R. A. (2007). Plasma antioxidant capacit y changes following a meal as a measure of the ability of a food to alter in vivo antioxidant status. J Am Coll Nutr, 26(2), 170 181. Prior, R. L., Hoang, H., Gu, L., Wu, X., Bacchiocca, M., Howard, L., Hampsch Woodill, M., Huang, D., Ou, B., & Jacob, R. ( 2003). Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance capacity (ORACFL)) of plasma and other biological and food samples. Journal of Agricultural and Food Chemistry, 51(11), 3273 3279. Prior, R. L., Lazarus, S. A., Ca o, G., Muccitelli, H., & Hammerstone, J. F. (2001). Identification of Procyanidins and Anthocyanins in Blueberries and Cranberries (Vaccinium Spp.) Using High Performance Liquid Chromatography/Mass Spectrometry. Journal of Agricultural and Food Chemistry, 49(3), 1270 1276. Prior, R. L., Wu, X., Gu, L., Hager, T., Hager, A., Wilkes, S., & Howard, L. (2009). Purified berry anthocyanins but not whole berries normalize lipid parameters in mice fed an obesogenic high fat diet. Mol Nutr Food Res, 53(11), 1406 141 8.

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78 Prior, R. L., Wu, X., Gu, L., Hager, T. J., Hager, A., & Howard, L. R. (2008a). Whole berries versus berry anthocyanins: interactions with dietary fat levels in the C57BL/6J mouse model of obesity. J Agric Food Chem, 56(3), 647 653. Prior, R. L., Wu, X. Gu, L., Hager, T. J., Hager, A., & Howard, L. R. (2008b). Whole berries versus berry anthocyanins: interactions with dietary fat levels in the C57BL/6J Mouse model of obesity. Journal of Agricultural and Food Chemistry, 56(3), 647 653. Rice Evans, C., & Miller, N. (1996). Antioxidant activities of flavonoids as bioactive components of food. Biochem. Soc. Trans,, 24, 790 795. Rodriguez Mateos, A., Cifuentes Gomez, T., Tabatabaee, S., Lecras, C., & Spencer, J. P. E. (2011). Procyanidin, Anthocyanin, and Chl orogenic Acid Contents of Highbush and Lowbush Blueberries. Journal of Agricultural and Food Chemistry. Sandhu, A. K., Gray, D. J., Lu, J., & Gu, L. (2011). Effects of exogenous abscisic acid on antioxidant capacities, anthocyanins, and flavonol contents o f muscadine grape (Vitis rotundifolia) skins. Food Chemistry, 126(3), 982 988. Seemann, J. R., & Sharkey, T. D. (1987). The effect of abscisic acid and other inhibitors on photosynthetic capacity and the biochemistry of CO2 assimilation. Plant Physiology, 84(3), 696 700. Sellappan, S., Akoh, C. C., & Krewer, G. (2002). Phenolic Compounds and Antioxidant Capacity of Georgia Grown Blueberries and Blackberries. Journal of Agricultural and Food Chemistry, 50(8), 2432 2438. Shulman, G. I. (2000). Cellular mechan isms of insulin resistance. Journal of Clinical Investigation, 106(2), 171 176. Sun, L., Zhang, M., Ren, J., Qi, J., Zhang, G., & Leng P. (2010). Reciprocity between abscisic acid and ethylene at the onset of berry ripening and after harvest. BMC Plant B iology, 10(1), 257. Tsuda, T., Horio, F., Uchida, K., Aoki, H., & Osawa, T. (2003). Dietary cyanidin 3 O D glucoside rich purple corn color prevents obesity and ameliorates hyperglycemia in mice. The Journal of Nutrition, 133(7), 2125 2130. Tsuda, T., Ue no, Y., Aoki, H., Koda, T., Horio, F., Takahashi, N., Kawada, T., & Osawa, T. (2004). Anthocyanin enhances adipocytokine secretion and adipocyte specific gene expression in isolated rat adipocytes. Biochemical and Biophysical Research Communications, 316(1 ), 149 157. USDA (2006). National nutrient database for standard reference. vol. 2010. USHBC (2012). Blueberry Nutrition Research.

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79 Vuong, T., Benhaddou Andaloussi, A., Brault, A., Harbilas, D., Martineau, L. C., Vallerand, D., Ramassamy, C., Matar, C., & Haddad, P. S. (2009). Antiobesity and antidiabetic effects of biotransformed blueberry juice in KKAy mice. Int J Obes, 33(10), 1166 1173. W. L McCabe J.C. Smith, P. H. (1993). Unit operations of chemical engineering. Singapore: McGrawHill. Walton, D. C. ( 1980). Biochemistry and physiology of abscisic acid. Annual Review of Plant Physiology, 31, 453 489. Worch, R. K. E. (1990). Adsorption aus wa¨ssrigen Lo¨sungen; VBE Deutscher Verlag fur Grundstoffindustrie. Leipzig, Germany. Wu, X., Beecher, G. R., Holden, J. M., Haytowitz, D. B., Gebhardt, S. E., & Prior, R. L. (2004). Lipophilic and Hydrophilic Antioxidant Capacities of Common Foods in the United States. Journal of Agricultural and Food Chemistry, 52(12), 4026 4037. Wu, X., Kang, J., Xie, C., Burri s, R., Ferguson, M. E., Badger, T. M., & Nagarajan, S. (2010). Dietary Blueberries Attenuate Atherosclerosis in Apolipoprotein E Deficient Mice by Upregulating Antioxidant Enzyme Expression. The Journal of Nutrition, 140(9), 1628 1632. Wu, X., & Prior, R. L. (2005). Systematic Identification and Characterization of Anthocyanins by HPLC ESI MS/MS in Common Foods in the United States: Fruits and Berries. Journal of Agricultural and Food Chemistry, 53(7), 2589 2599. Xu, Z., Zhang, Q., Chen, J., Wang, L., & And erson, G. K. (1999). Adsorption of naphthalene derivatives on hypercrosslinked polymeric adsorbents. Chemosphere, 38(9), 2003 2011. Youdim, K. A., McDonald, J., Kalt, W., & Joseph, J. A. (2002). Potential role of dietary flavonoids in reducing microvascula r endothelium vulnerability to oxidative and inflammatory insults. The Journal of Nutritional Biochemistry, 13(5), 282 288. Zhang, M., Leng, P., Zhang, G., & Li, X. (2009). Cloning and functional analysis of 9 cis epoxycarotenoid dioxygenase (NCED) genes e ncoding a key enzyme during abscisic acid biosynthesis from peach and grape fruits. Journal of Plant Physiology, 166(12), 1241 1252. Zhang, M., Yuan, B., & Leng, P. (2009). The role of ABA in triggering ethylene biosynthesis and ripening of tomato fruit. J ournal of Experimental Botany, 60(6), 1579 1588. Zheng, W., & Wang, S. Y. (2002). Oxygen radical absorbing capacity of phenolics in blueberries, cranberries, chokeberries, and lingonberries. Journal of Agricultural and Food Chemistry, 51(2), 502 509.

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80 BIOGRAPHICAL SKETCH Tim Buran graduated from the University of Illinois Urbana Champaign with a Bachelor of Science degree in Food Science and Human Nutrition in 2009. From there, he went out west to San Francisco where he worked as a product development s cientist for Del Monte Foods. In the summer of 2010, Tim accepted a position with Dr. Liwei Gu. This summer, Tim will be graduating with is m Nutrition. He will leave with three publications and two invitations to present a t IFT including this year where he was selected as a finalist in the Gradu ate Research Paper Competition. Upon the completion of his degree, Tim ho pes to return to industry as a p roduc t d evelopmen t s cientist.