ABSTRACT Title of Document: INCORPORATING RAPE SEED ANTIOXIDANTS INTO A FUNCTIONAL FOOD MODEL Lena Binzer Rebeca Brinsko Jessica Cha Zao Chen Sarah Green Kelly Grob Junjie Hao Christina Hitz Laura Li Sowmya Swamy Maxim Wolf MengMeng Xu ary Yanik Directed By: Dr. Liangli Yu, PhD, Department of Nutrition and Food Science Dr. Margaret Slavin, PhD, RD, Department of Nutrition and Food Science Consumption of foods rich in natural antioxidants may potentialy reduce the risk of chronic ilneses. This study examined feasibility and consumer aceptability of creating a functional food rich in natural antioxidants from cold-presed grape seed oil and flour. The first study investigated and compared five grape sed varieties, and found Chardonnay grape seds contained the highest quantity of health-beneficial properties. Consequently, addition of Chardonnay grape seed flour and oil to bread significantly increased health-beneficial properties. Baking conditions influenced antioxidant properties of bread, indicating procesing conditions may affect antioxidant activity in finished food products. In addition, the consumer sensory evaluation study found control bread was preferred over bread containing grape sed flour and oil; however, grape sed containing bread had an overal positive reception. Incorporation of grape seds into bread may be efective in incorporating health-beneficial compounds into the diet; however, further studies on long term health effects should be conducted. INCORPORATING RAPE SEED ANTIOXIDANTS INTO A FUNCTIONAL FOOD MODEL By Team Innovative Medicines for Maladies Utilizing Nutraceutical Enhancements (IMMUNE) Lena Binzer, Rebeca Brinsko, Jessica Cha, Zao Chen, Sarah Green, Kelly Grob, Junjie Hao, Christina Hitz, Laura Li, Sowmya Swamy, Maxim Wolf, MengMeng Xu, & Mary Yanik Thesis submited in partial fulfilment of the requirements for the Gemstone Program, University of Maryland, College Park 2011 Advisory Commite: Dr. Liangli Yu, Mentor Dr. Margaret Slavin, Mentor Dr. William J. Kenworthy, Discussant Dr. Jeffrey Moore, Discussant Dr. Qin Wang, Discussant Dr. Thomas Wang, Discussant ? Copyright by Team IMMUNE Lena Binzer, Rebeca Brinsko, Jessica Cha, Zao Chen, Sarah Green, Kelly Grob, Junjie Hao, Christina Hitz, Laura Li, Sowmya Swamy, Maxim Wolf, MengMeng Xu, & Mary Yanik 2011 ii Acknowledgements Our research over the past four years would not have been possible without the contribution and support from various members of industry, the scientific community, as well as the University of Maryland. We have many people to thank for their contributions towards the succes of our project. Foremost, we would like to expres our gratitude towards our research mentor, Dr. Liangli Yu, and co-mentor Dr. Margaret Slavin and our past co-mentor Dr. Mickey Parish, for their guidance, encouragement and support over the past 3.5 years. We would especialy like to thank Dr. Yu for the extensive use of her laboratory, as well as her group members for their asistance and mentorship in obtaining and interpreting experimental results. We would also like to thank our commite members, Dr. William Kenworthy, Dr. Jefrey Moore, Dr. Qin Wang, and Dr. Thomas Wang, for the time they took to provide constructive and detailed comments on our disertation and expanding our vision of the project through their fedback. Many thanks to the Gemstone Program staff, for their insight and support of our project, from its conceptualization to its realization. Their continuing fedback has helped to shape and improve the project over the past 4 years. We would like thank our family and friends for their support and encouragement, which have motivated us through dificult times throughout our project. Financial support for this research was provided by ACC International Academic Collaborative Felows Program, Maryland Grain Producers Utilization Board, Maryland Soybean Board, Botanical Oil Innovations Inc., and the University of Maryland Gemstone Program. In addition, material support in the form of whole wheat flour, and cold presed iii grape seed flour and oil were provided by the Mennel Miling Company (Fostoria, OH) and Botanical Oil Innovations Inc. (Spooner, WI), respectively. Their tremendous contribution to the succes of Team IMMUNE is gratefully acknowledged. iv Table of Contents Acknowledgements.......................................................................................................ii! List of Tables..............................................................................................................vii! List of Figures............................................................................................................vii! Chapter 1: Introduction..................................................................................................9! Introduction....................................................................................................................9! Chapter 2: Antioxidant Properties and Phenolic Components of Grape Seds...........14! Introduction..................................................................................................................14! Phenolic Components..................................................................................................15! Total Phenolic Content................................................................................................15! Phenolic Composition of Grape Seds........................................................................24! Trans-Resveratrol........................................................................................................30! Antioxidant Properties.................................................................................................31! Antioxidative Properties of Grape Seds.....................................................................31! Efects of Post-Harvest Treatments on Grape Sed Antioxidants...............................38! Efects of Thermal Treatment......................................................................................38! Efects of Particle Size.................................................................................................43! Efects of Storage Conditions......................................................................................43! Considerations in Antioxidant Property Estimation for Grape Seds.........................44! Summary......................................................................................................................45! Incorporation Into Functional Foods...........................................................................45! Functional Foods: An Overview..................................................................................45! Antioxidants in Functional Foods................................................................................46! Bread as a Functional Food Model..............................................................................48! Additives in Bread Models..........................................................................................49! Antioxidants.................................................................................................................49! Faty Acids...................................................................................................................50! Fiber.............................................................................................................................50! Diet and Health Efects................................................................................................52! Supplementation..........................................................................................................52! Functional Foods.........................................................................................................56! Summary......................................................................................................................57! Chapter 3: Chemical Composition and Health Properties of the Selected Cold-Presed Grape Sed Flours and Oils.........................................................................................59! Introduction..................................................................................................................59! Materials and Methods................................................................................................61! aterials and Chemicals..............................................................................................61! Lipid Extraction...........................................................................................................61! Antioxidant Extraction.................................................................................................62! Faty Acid Composition...............................................................................................62! Carotenoid/Tocopherol Composition..........................................................................63! Total Phenolic Content................................................................................................64! v Oxidative Stability Index (OSI)...................................................................................64! Oxygen Radical Absorbance Capacity (ORAC) Asay...............................................65! Hydroxyl Radical Scavenging Capacity (HOSC) Estimation.....................................65! DPH? Scavenging Activity........................................................................................66! Anti-proliferative Activity against HT-29 Cels..........................................................66! Statistical Analysis.......................................................................................................67! Results and Discussion................................................................................................68! Faty Acid Composition...............................................................................................68! Carotenoid/Tocopherol Composition..........................................................................69! Total Phenolic Content................................................................................................72! Oxidative Stability Index (OSI)...................................................................................74! Antioxidant Properties.................................................................................................76! Anti-proliferative Activity against HT-29 Cels..........................................................81! Conclusion...................................................................................................................84! Chapter 4: Chemical Composition and Health Properties of Whole Soft Wheat Bread Enriched with Cold-Presed Chardonnay Grape Sed Flour and Oil..........................85! Introduction..................................................................................................................85! Materials and Methods................................................................................................87! aterials and Chemicals..............................................................................................87! Baking Proces.............................................................................................................87! Lipid Extraction...........................................................................................................88! Antioxidant Extraction.................................................................................................88! Lutein/Tocopherol Composition..................................................................................89! Total Phenolic Content................................................................................................89! Oxygen Radical Absorbance Capacity........................................................................90! Hydroxyl Radical Scavenging Capacity......................................................................91! DPPH ? Scavenging Activity.........................................................................................91! Anti-Proliferative Activity against HT-29 Cels..........................................................92! Statistical Analysis.......................................................................................................93! Results and Discussion................................................................................................93! Lutein and "-Tocopherol Composition.......................................................................93! Total Phenolic Content................................................................................................95! Antioxidant Properties.................................................................................................97! Anti-Proliferative Activity against HT-29 Cels........................................................102! Conclusion.................................................................................................................107! Chapter 5: Consumer Aceptability of Functional Bread Containing Cold-presed Grape Sed Oil and Flour..........................................................................................108! Introduction................................................................................................................108! Methods.....................................................................................................................110! Sample Preparation....................................................................................................110! Sensory Analysis.......................................................................................................111! Data Analysis.............................................................................................................113! Results........................................................................................................................114! Conclusion.................................................................................................................117! Chapter 6: Conclusion..............................................................................................119! Limitations.................................................................................................................121! vi References..................................................................................................................124! Appendices................................................................................................................144! Appendix A: Script....................................................................................................144! Appendix B: Consent Form.......................................................................................146! Appendix C: Team IMMUNE Survey......................................................................148! Glossary.....................................................................................................................149! vii List of Tables Table 2. 1: Total phenolic, flavanol, and anthocyanin contents of grape seds..........19 Table 2. 2: Total phenolic content, anthocyanin content, and antioxidant capacity of whole fruit and parts of Muscadine grapes..................................................................21 Table 2. 3: Polyphenolic composition of grape seds.................................................26 Table 2. 4: Polyphenolic composition of seds and skins of red and white grape varieties........................................................................................................................28 Table 2. 5: Efects of thermal treatment on total phenolic content (mM TAE) of whole and powdered grape (V. vinifera, Campbel early) seds.................................39 Table 2. 6: Efects of diferent extraction times and crushing on total phenolic, anthocyanin, and flavanol contents of grape sed extracts..........................................42 Table 3. 1: Faty Acid (FA) composition of the studied cold-presed grape sed flours (g/100 g oil).................................................................................................................69 Table 5. 1: Mean preference ratings of color, texture, and flavor for grape sed and control breads.............................................................................................................116 Table 5. 2: Color, texture, and flavor mean preference interval at 95% confidence level for grape sed and control breads.....................................................................116 vii List of Figures Figure 2. 1: Phenolic components of grape seds........................................................17 Figure 2. 2: Oxygen radical absorbance capacity (ORAC) of grape seed extracts.....32 Figure 2. 3: Antioxidant activity of grape seed and skin of diferent grape varieties native to Tuscan...........................................................................................................34 Figure 2. 4: Inhibitory effects of individual grape phenolic compounds on lipid peroxidation in human LDL........................................................................................36 Figure 2. 5: Effects of thermal treatment and particle size on antioxidant stability in whole and powdered grape seds................................................................................41 Figure 3. 1: Composition of !-tocopherol and carotenoids in grape sed oils............71 Figure 3. 2: Total phenolic contents of grape sed flours............................................74 Figure 3. 3: Oxidative stability index of grape sed oils.............................................76 Figure 3. 4: Hydroxyl radical scavenging capacity of grape sed flours.....................77 Figure 3. 5: Oxygen radical scavenging capacity of grape sed flours.......................78 Figure 3. 6A: DPH radical scavenging capacity of grape sed flours.......................80 Figure 3. 6B: DPH radical scavenging capacity of grape sed oils...........................80 Figure 3. 7: Time and dose efects of grape sed flour and oil extracts on HT-29 cel proliferation.................................................................................................................80 Figure 4. 1: Lutein and !-tocopherol content of control and grape seed breads based on baking conditions....................................................................................................94 Figure 4. 2: Total phenolic content of control and grape sed breads based on baking conditions.....................................................................................................................96 Figure 4. 3: Oxygen radical absorbance capacity of control and grape sed breads based on baking conditions..........................................................................................99 Figure 4. 4: Hydroxyl radical scavenging capacity of control and grape sed breads based on baking conditions........................................................................................100 Figure 4. 5: DPH radical scavenging capacity of control and grape sed breads based on baking conditions..................................................................................................102 Figure 4. 6: Anti-proliferative efects of grape sed bread based on baking temperature................................................................................................................104 Figure 4. 7: Anti-proliferative efects of grape sed bread based on baking time.....105 Figure 4. 8: Proliferation of cels treated with control and grape sed bread based on baking conditions.......................................................................................................106 Figure 5. 1: Sensory analysis set-up..........................................................................112 Figure 5. 2: Box and whisker plot of grape sed sensory analysis data....................117 9 Chapter 1: Introduction Introduction Chronic diseases, such as heart disease, cancer, obesity, and diabetes, are currently the prominent cause of death in the United States. The Centers for Disease Control and Prevention (CDC) estimates that 7 out of 10 deaths among Americans each year are from chronic diseases (CDC, 2010). These long-term ilneses carry a substantial financial burden. Acording to the CDC, they are responsible for three dollars out of every four dollars that is spent on healthcare in the United States (CDC, 2005). The impact of these types of ilneses is not restricted to developed nations. The World Health Organization (WHO) reports that chronic ilneses cause roughly 60% of all deaths globaly (CDC, 2011). Significant research has been devoted to finding novel methods for the prevention and treatment of chronic disease. In 1992, research sought to identify the relatively low incidence of chronic diseases in France as opposed to in the United States despite the French diet being shown to be similar to American diet rich in saturated fats typicaly asociated with promotion of cardiovascular disease. This phenomenon was termed the French Paradox (de Lange, 2007). The French Paradox led to increased research regarding the diferences betwen the diets and lifestyles of the French and American people; the most striking distinction was the fact that the French were acustomed to consuming wine daily (Renaud & de Lorgeril, 1992). More recent studies have shown that chemical compounds found in grapes and wine, such as resveratrol, offered a possible explanation to the French Paradox (Dulak, 2005), and reinforced the importance of diet in the 10 prevention of chronic disease. Resveratrol is just one of many polyphenolic antioxidants found in grapes and grape seds and will be discussed in further detail below. Genetic predisposition and lifestyle/environment choices are the two main risk factors for chronic ilneses. Since innate genetic traits cannot be altered, eforts toward the prevention of long-term ilnes focus on improving lifestyles. Risks for developing chronic ilneses has been linked to oxidative stres, which has multiple causes, including overexposure to sunlight, smoking, daily exposure to certain chemicals, and improper diet. The human body functions through carefully monitored oxidation-reduction reactions, both inside and outside the cells. Oxidative stres can shift this delicate balance, resulting in an elevated level of free radicals in the body (Hennig et al., 2007). Free radicals are very reactive chemical species that exist in the human body as a natural product of metabolism and function. Sustained elevated levels of oxygen centered free radicals experienced during oxidative stres can lead to uncontrolled and undesired reactions that damage the macromolecules in cells, including proteins, lipids, and DNA. Damage to these molecules can result in an increased risk of cancer and cardiovascular disease (Seifried et al., 2007). Dietary choices play an important role in disease prevention. It is known that certain fruits and vegetables contain phytochemicals, esential faty acids, traditional vitamins and minerals, and other bioactive compounds. However, the CDC released a report in 2005 stating that only 33% of American adults eat fruit two or more times daily and only 27% eat vegetables three or more times daily (Blanck et al., 2005). The phytochemicals of fruits and vegetables have been shown to reduce the risk of cancer and cardiovascular disease (Liu, 2003). There is growing interest in the health enhancing role of certain foods and food components. This has led to the development and use of the term ?functional foods.? Acording 11 to the Institute of Food Technologists (IFT), a ?functional food? is considered a food that provides an additional physiological benefit beyond that of its inherent nutritional value (Bidlack et al., 2005). Functional foods can be specificaly designed to incorporate antioxidants, which are chemicals with the ability to scavenge free radicals and reduce oxidative stres in the body (Eastwood, 1999). As opposed to pharmaceuticals, they also have the potential to be produced economicaly and made acesible to a large number of people. Grape seds can be exploited for functional food development, as they contribute health beneficial properties and are a byproduct of the juice and wine-making industries. Most antioxidants found in grape seed extracts are polyphenolics (Shi et al., 2003). The key types of phenolics present in grapes and grape seds are proanthocyanidins, anthocyanins, and resveratrol (Bozan et al., 2008; Lafka et al., 2007; Yilmaz & Toledo, 2004). Studies have shown that proanthocyanidins have preventative effects against cancer, heart disease, and aging by inhibiting malignant cell growth, delaying heart cell death, and maintaining membrane integrity as cells age (Prior & Gu, 2005; Karthikeyan et al., 2007; Nandakumar et al., 2008). Anthocyanins are the pigments which give grapes and grape seds their red and purple color. Anthocyanins have been reported to exhibit health-promoting properties, including anti- inflamation of blood vesels and reduction of platelet coagulability, which may reduce risks of developing atherosclerosis, an initial step in cardiovascular disease (Maza, 2007). Studies have shown that resveratrol, as well as anthocyanins, may positively affect heart health by modulating proteins regulating imune responses, including inflamation of the vascular system (Falcheti et al., 2001; Tao et al., 2004). Overal, polyphenolics appear to be potentialy capable of preventing cancer and cardiovascular diseases. 12 In addition to polyphenolics, antioxidants found in grape seds include certain vitamins, specificaly vitamin C (ascorbic acid) and vitamin E (tocopherols). Vitamins C and E, especialy when tested in combination, have been shown to positively affect the health of those with heart disease and diabetes. Vitamin E has been reported to increase the eficiency of cholesterol scavenging by monocytes. This property may be linked to the prevention of atherosclerosis, or the acumulation of plaque lining blood vesel walls, that causes many cardiovascular health problems (Cachia et al., 1998). Also, treatments utilizing vitamin C and E supplements aleviated the symptoms of Type II diabetes patients (Chui & Greenwood, 2008). Grape seds are also a source of healthy faty acids and dietary fibers (Cao & Ito, 2003). Some unsaturated faty acids contained in grape seed oil, such as !-linolenic acid ("-3) and #- linolenic acid ("-6), are considered esential faty acids because they cannot be produced by humans (Smith, 2007). Consumption of unsaturated faty acids has been correlated to a reduction of cardiovascular disease, cancer, hypertension, and autoimune disorders (Aronson et al., 2001). Additionaly, dietary fiber makes up a considerable portion of grape seds and peels? about 80% of their dry weight?and has been linked with lower risks of heart disease, obesity, diabetes, and colon cancer (Go?i et al., 2005). The addition of grape seds to a staple food may increase the quantity of fiber in the diet. Grape seds are produced en masse as a byproduct from wine and juice production industries. However, their disposal has been shown to negatively impact to the environment. Grape seed waste has high concentrations of organic (carbon-rich) substances. The decomposition of this material decreases the amount of oxygen available to other organisms, resulting in detrimental effects on the ecosystem where they are disposed (Lafka et al., 2007). Therefore, if grape seds were adopted as a functional food ingredient on a commercial scale, the 13 potential exists to raise the agricultural value of grapes and significantly decrease the amount of waste produced by the wine and juice industries, minimizing the negative environmental impacts of grape procesing. The purpose of Team IMMUNE?s (Innovative Medicines for Maladies Utilizing Nutraceutical Enhancements) project is to increase the intake of health-beneficial compounds in the American diet without dietary supplements. We explored the feasibility of incorporating the antioxidant properties of grape seed by-products from the wine and juice industry into bread, a staple food in the American diet, in acordance with the IFT definition of a functional food. Specificaly, cold-presed (temperature controlled procesing method) grape seed flours and oils were used. The ultimate impact of our project could provide a convenient avenue for reducing medical costs incurred by chronic ilneses and introduce a new use for otherwise wasteful by- products of a large industry. Our study has the added benefit of increasing economic value of grape seds, which are currently disposed of as a waste product of wine and juice production. Our project had three main research objectives. First, we measured the health-benefical properties of five varieties of cold-presed grape seed flours and oils (Chardonnay, Concord, Norton, Ruby Red, and White) to determine which one had the highest potential for development of a model functional food. We then incorporated the seed flour and oil of this variety, Chardonnay, into a bread product and determined the optimal baking temperature, time, and flour composition necesary to produce a viable final bread product that retains the most health- beneficial properties. Finaly, we conducted an institutional review board (IRB) approved sensory analysis of our product on freshman Gemstone students at the University of Maryland College Park Campus. 14 Chapter 2: Antioxidant Properties and Phenolic Components of Grape Seeds Introduction Grapes are one of the largest fruit crops in the world, with approximately 66 milion tons produced worldwide in 2007, and over 6.1 milion tons produced in the United States alone (FAOSTAT, 2007). Approximately 86.6% of fresh grapes are procesed to produce wine, jams, and grape juice (Maier et al., 2009). Seds comprise 5% by-mass of grapes and are a primary by- product from grape procesing industries. Grape seds are composed of 10-20% oil, along with fiber, protein, and other components, including phenolic antioxidants (Kim et al., 2006; Choi & Lee 2008). Investigation into the health beneficial properties such as antioxidative capacity is important for development of value-added utilization of grape seds (Parry et al., 2006). A number of studies have investigated antioxidant components of grape seds and seed fractions (Luther et al., 2007). Antioxidant properties of grape seds, seed fractions, and individual grape antioxidant compounds have also been evaluated. This review summarizes the available information on phenolic components in grape seds, their antioxidant properties, effects of post- harvest treatments on grape antioxidants, and analytical considerations for grape antioxidant property estimation. 15 Phenolic Components Total Phenolic Content Grape seds are rich in phenolic compounds such as anthocyanins, the glucosides of anthocyanidins (Fig. 2.1A), catechin (Fig. 2.1B), and galic acid (Fig. 2.1C). Figure 2.1A Anthocyanidin R1 R2 Cyanidin OH H Delphinidin OH OH Malvidin OMe OMe Pelagonidin H H Petunidin OMe OH Peonidin OMe H 16 Catechin Epicatechin Catechin Epicatechin R1 R2 (+)-Catechin (-)-Epicatechin H H (+)-Galocatechin (-)-Epigalocatechin OH H (+)-Catechin-3-galate (-)-Epicatechin-3-galate H Gallate (+)-Galocatechin-3-galate (-)-Epigalocatechin-3-galate OH Gallate Figure 2.1B 17 Figure 2.1C Figure 2. 1 Chemical structures of grape phenolic compounds. (A) Anthocyanidins; (B) Catechins (structure on the left is a (+)-catechin skeleton, while the structure on the right is an (-)- epicatechin skeleton); and (C) Quercetin, gallic acid, and trans-resveratrol. Quercetin Galic Acid trans-Resveratrol 18 Polyphenolics generaly contain two or more hydroxyl groups atached to a conjugated ring system such as a benzene ring. Polyphenols contribute to the overal antioxidant properties of grapes and may have important health benefits, including possible preventative effects against cancer (Fan & Lou, 2004) and cardiovascular diseases (Zern et al., 2003; Zern et al., 2005). Anthocyanins have been shown to protect against lipid peroxidation and DNA damage in rat hepatoma cells in vitro, demonstrating potential anticarcinogenic properties (Laz? et al., 2003). Flavanols, such as catechin, have demonstrated inhibitory effects on platelet reactivity in vitro, a property which may reduce the risk of cardiovascular disease (Pearson et al., 2005). Gallic acid has been shown to exhibit selective cytotoxicity in vivo against a variety of human and mouse cancer cells (Inoue et al., 1995). Additionaly, grape seed extracts rich in polyphenolics have been found to reduce biomarkers of Alzheimer?s in a rat model (Thomas et al., 2009). This result was supported by a separate finding suggesting that catechins from grape seed extracts fed in a controlled diet to rats were able to cross the blood-brain barrier, as evidenced by heightened levels of catechin and epicatechin detected in the brain of rats fed the grape seed extract enriched diet (Prasain et al., 2009). Table 2.1 summarizes the total phenolic (TPC), total flavanol (TFC), and total anthocyanin (TAC) contents of selected grape varieties estimated using spectrophotometric methods from previous studies. 19 Table 2. 1 Total phenolic, flavanol, and anthocyanin contents of grape seeds. Variety TPC (mg GAE/g) TFC (mg/g) TAC (mg CGE/10 g) Reference Ebizuru 8.8 F 1.3 QE F ND Poudel et al., 2008 Ryukyuganebu 3.6 F 1.0 QE F ND Poudel et al., 2008 Shiohtashibudou 13.6 F 5.5 QE F ND Poudel et al., 2008 Shiragabudou 16.5 F 1.4 QE F ND Poudel et al., 2008 Yamabudou 5.7 F 0.8 QE F ND Poudel et al., 2008 Kadainou R-1 8.7 F 0.9 QE F ND Poudel et al., 2008 Kadainou R-1 x Bailey Alicante A. 8.7 F 0.7 QE F ND Poudel et al., 2008 Bailey Alicante A 17.9 F 0.9 QE F ND Poudel et al., 2008 Muscat of Alexandria 54.9 F 1.0 QE F ND Poudel et al., 2008 Merlot 105.7 D 122.7 CE D NA Bozan et al., 2008 Cabernet 103.7 D 125.0 CE D NA Bozan et al., 2008 Cinsault 88.1 D 97.1 CE D NA Bozan et al., 2008 Papaz Karasi 154.6 D 179.4 CE D NA Bozan et al., 2008 Ada Karasi 137.5 D 163.4 CE D NA Bozan et al., 2008 Hamburg Muscat 104.4 D 105.7 CE D NA Bozan et al., 2008 Alphonso Lavale 105.3 D 123.3 CE D NA Bozan et al., 2008 Okuzgozu 139.4 D 174.5 CE D NA Bozan et al., 2008 Bogazkere 94.2 D 95.0 CE D NA Bozan et al., 2008 Senso 79.2 D 89.2 CE D NA Bozan et al., 2008 Kalecik Karasi 136.2 D 147.7 CE D NA Bozan et al., 2008 Bronze Muscadine 19.2-32.6 a F NA 1.2-8.7 a F Pastrana-Bonila et al., 2003 Purple Muscadine 15.4-26.9 b F NA 2.2-7.5 b F Pastrana-Bonila et al., 2003 Cabernet Sauvignon 8.7 F NA NA Guendez et al., 2005 Grenache Rouge 9.8 F NA NA Guendez et al., 2005 Merlot 16.9 F NA NA Guendez et al., 2005 Mandilaria 22.3 F NA NA Guendez et al., 2005 Agiorgitiko 11.3 F NA NA Guendez et al., 2005 Negoska 11.8 F NA NA Guendez et al., 2005 Xinomavro 1.4 F NA NA Guendez et al., 2005 Mavrodafni 4.0 F NA NA Guendez et al., 2005 Limnio 14.1 F NA NA Guendez et al., 2005 a : range for five diferent varieties of bronze Muscadine grapes; b : range for five diferent purple Muscadine grapes. TPC: total phenolic content; TFC: total flavanol content; TAC: total anthocyanin content; GAE: galic acid equivalent; QE: quercetin equivalent; CE: catechin equivalent; CGE: cyanidin-3-glucoside equivalent; D : dry weight; F : fresh weight; NA: not analyzed; ND: not detected. 20 In general, TPC values were determined colorimetricaly using Folin-Ciocalteu (FC) reagent. However, this method measures total reducing capacity, and thus may also measure other non-phenolic reducing agents, such as ascorbic acid. Of the grape varieties analyzed, Papaz Karasi seds contained the highest total phenolic content of 154.6 mg galic acid equivalents (GAE)/g on a per dry seed weight basis, followed by Okuzgozu at 139.4 mg GAE/g, and Ada Karasi at 137.5 mg GAE/g (Bozan et al., 2008). Of the grape seds reported on a per fresh weight basis, the seds of Muscat of Alexandria contained the highest total phenolic content of 54.9 mg GAE/g seed (Poudel et al., 2008), followed by 32.6 mg GAE/g in Summit Muscadine (bronze) and 26.9 mg GAE/g in Noble Muscadine (purple) grape seds (Pastrana-Bonila et al., 2003). Under the same experimental conditions, the pulp, skin, and whole fruit of Summit Muscadine grape (Table 2.2) contained about 0.22, 5.41, and 3.10 mg GAE/g on a fresh weight basis, respectively, which is less than 1, 20, and 10% of that in the seds (Pastrana-Bonila et al., 2003). Pastrana-Bonila et al., (2003) analyzed the TPC of ten diferent varieties of Muscadine grapes, five with bronze skins and five with purple skins. The seds of the five bronze grapes had TPC value of 19.2-32.6 mg GAE/g, which was much higher than that of 3.0-5.5, 0.21-0.25, and 1.7-3.1 mg GAE/g determined in the skin, pulp, and whole fruit, respectively (Table 2.2). The total phenolics in the seds were, on average, five times more concentrated than that in the skin and 80 times more concentrated than that in the pulp on a fresh weight basis (Table 2.2), suggesting that grape seds may serve as an excelent source for dietary phenolic components. Notably, for the phenolics to be available to humans, the seds must be ground prior to consumption in order to expose them to digestion; otherwise, whole seds pas through largely undigested. 21 Table 2. 2 Total phenolic content, anthocyanin content, and antioxidant capacity of whole fruit and parts of Muscadine grapes. a TPC (mg GAE/100 g) TAC (mg CGE/100 g) TEAC (!M/g) Variety Seed Skin Pulp Whole Fruit Seed Skin Pulp Whole Fruit Seed Skin Pulp Whole Fruit Bronze Carlos 1920.3 545.6 25.1 307.9 1.2 2.6 ND 0.9 204.6 14.9 3.4 18.2 Early Fry 2367.2 303.0 21.3 169.1 8.7 2.5 ND 1.1 277.8 13.9 2.0 11.2 Fry 2356.3 332.2 23.8 199.0 4.6 0.8 ND 0.4 234.2 11.1 2.9 9.8 Summit 3258.7 541.0 22.3 309.7 3.1 2.8 ND 1.3 245.4 12.4 3.0 10.2 Late Fry 1986.0 348.9 24.0 252.3 3.7 2.0 ND 1.1 218.9 13.4 2.4 15.4 AV 2377.7 414.1 23.3 247.6 4.3 2.1 ND 1.0 236.2 13.1 2.7 13.0 Purple Paulk 1649.3 363.6 30.0 195.2 4.1 177.0 4.7 74.8 307.9 12.1 2.2 11.2 Cowart 2303.0 261.6 11.6 214.2 4.6 107.8 1.1 37.8 325.5 12.4 2.7 21.7 Supreme 1535.5 329.9 20.1 184.7 7.5 135.5 0.7 65.2 478.6 12.2 1.6 11.5 Ison 1726.2 365.0 26.0 218.9 4.6 174.5 1.9 69.5 284.9 13.3 2.1 15.9 Noble 2685.3 355.1 33.4 425.7 2.2 65.5 2.2 31.5 234.7 12.4 2.1 27.8 AV 1979.9 335.0 24.2 247.7 4.6 132.1 2.1 55.8 326.3 12.5 2.1 17.6 a Data from Pastrana-Bonila et al. 2003 TPC: total phenolic content; TAC: Total anthocyanin content; TEAC: trolox equivalent antioxidant capacity; GAE: galic acid equivalent; CGE: cyanidin 3-glucoside equivalent; ND: not detected; AV: average value 22 The TPC of grape seed flours, oils, and extracts have been investigated. In 2006, Parry and others reported the TPC value of various fruit seed flours, including that of Pinot Noir and Chardonnay grape varieties. Chardonnay seed flour exhibited the highest TPC with 186.3 mg GAE/g flour, which was higher than that of Pinot Noir, and also higher than that of black and red raspberry, blueberry, and cranberry seed flours (Parry et al., 2006). Bail et al., analyzed the TPC values of nine European grape seed oils, and found that the highest TPC value was 0.1 mg GAE/g seed oil whereas the lowest was 0.06 mg GAE/g oil, suggesting that grape seed oils may not be a good source of phenolic compounds (2008). TPC of seed extracts of several Turkish grape varieties were evaluated by Yemis and others (2008) and Narince grape seed extract was shown to contain the highest total phenolic content with 587.3 mg GAE/g extract, while the lowest TPC value was 339.5 mg GAE/g extract. In 2009, using HPLC, Maier and others determined TPC of the intact grape seds, seed oil pres residues, and the seed flour which is the byproduct of grape seed-oil extraction of seven varieties of Vitis vinifera. The seds had TPC values ranging from 188.7 to 1165.8 mg/kg dry matter (DM), which was statistically higher than the corresponding values for the seed flour, ranging from 147.4 to 492.7 mg/kg DM, suggesting that grape seed oil may contain significant levels of phenolic compounds, while the seed flour may also serve as a dietary source of phenolics (Maier et al., 2009). The highest reported total flavanol content (TFC) per fresh weight was found in Shiohtashibudou grape seds, at a level of 5.5 mg quercetin equivalents (QE)/g (Poudel et al., 2008), while Papaz Karasi contained the highest flavanol content on per dry seed weight basis, with 179.4 catechin equivalents (CE)/g (Bozan et al., 2008). Table 2.1 provides a summary of representative TFC values of grape seds from several previous studies. The seds, skins, and pulp of Pinot Noir, Pinot Meunier, and the Chardonnay grape were analyzed with HPLC (Man? 23 et al., 2007). The results showed that seed of the Pinot Noir, Pinot Meunier, and Chardonnay grapes had TFC values of 75.1, 101, and 57.6 mg/g, respectively, on a per fresh weight basis, which were much greater than that of 30, 24, and 21 mg/g detected in the corresponding skin fractions, or that of 0.45, 0.26, and 0.36 mg/g in the corresponding pulp samples (Man? et al., 2007). It needs to be pointed out that these TFC values are estimated using catechin, epicatechin, epigalocatechin, or epicatechin-3-galate as a standard reference compound (Man? et al., 2007). In addition, Maier et al., compared the TFC of seven grape seds and their corresponding seed flours, the residue from seed oil pres, and showed that the seds contained 4.39-18.78 g TFC/kg and the seed flours had 2.5-13.5 g TFC/kg on per dry mass basis. The seed flour retained about 57-78.6% TFC after seed oil production, indicating that both whole seds and seed flour, the by- product from seed oil preparation, may serve as a dietary source of flavanoids (2009). Total anthocyanin content (TAC) has been quantified for grape seds in a number of previous studies using a UV-visible spectrophotometer acording to a pH-diferential method. The highest reported total anthocyanin content (TAC) was found in Early Fry Muscadine (bronze) grape seds (Table 2.2), with 8.7 mg cyanidin-3-glucoside equivalents (CGE)/100 g fresh seds (Pastrana-Bonila et al., 2003). It was interesting that seds of bronze Muscadine grapes had a TAC value ranging from 1.2 to 8.7 mg CGE/100 g fresh seds, whereas that of purple grapes had a TAC range of 2.2-7.5 mg CGE/100 g fresh seds (Pastrana-Bonila et al., 2003), suggesting that that TAC values of grape seds could not be predicted by the pigmentation in grape skins, although anthocyanin is the pigment responsible for the purple color in both grapes and wine. The data, in Table 2.2, show that there were low levels of anthocyanins in the seed and no anthocyanins detected in the pulp of the bronze varieties of Muscadine grapes. The anthocyanin content was only slightly higher in the skins of these varieties on a fresh weight 24 basis (Table 2.2). In the purple-skinned varieties of Muscadine grapes, the seds and pulp contained lower levels of anthocyanin at 2.2-7.5 and 0.7-4.7 mg CGE/100 g respectively, but the skins had high concentrations ranging from 65.5 to 177.0 mg CGE/100 g on a per fresh sample weight basis (Pastrana-Bonila et al., 2003). Total anthocyanin content in the seds of purple Muscadines grapes was, on average, 1.3 times higher than that of the bronze grapes, while the skins of purple varieties had about 65 times more anthocyanins than that of bronze varieties (Pastrana-Bonila et al., 2003). In summary, TPC values vary greatly in grape seds and seed fractions, suggesting that genotype and environmental conditions during growth may alter the phenolic component and contents in grape seds. For example, a recently published study by a group in China found that Asian varieties of grapes yielded, on average, seds and skins with lower levels of various phenolic compounds than European varieties, and that hybrids of the two yielded grape seds that fel within the range (Xu et al., 2010). In addition, a study reported in 2010 found that organicaly grown grapes contained higher levels of anthocyanins, flavanols, and total phenolic content than conventionaly grown grapes, suggesting that growing methods may also significantly affect the chemical composition of grapes, and therefore grape seds (Mulero et al., 2010). Overal, grape seds appear to be an excelent source of phenolic compounds, and can contribute significantly to the economic value of grapes for the viticulture industry. Phenolic Composition of Grape Seeds In addition to TPC, the quantities of individual polyphenolic compounds have also been of interest to researchers because they may contribute to the overal and selected health beneficial effects diferently. For example, they may have diferent antioxidant properties. 25 Catechins, anthocyanins, and other phenolic compounds such as galic acid have been detected in grape seds (Fig. 2.1) (Fuleki & da Silva, 1997; Kamerer et al., 2004; Guendez et al., 2005; Montealegre et al., 2006; Maier et al., 2009). Table 2.3 shows the concentrations of major polyphenolic compounds reported in grape seds; only a small part of the values from previous studies are included. 26 Table 2. 3 Polyphenolic composition of grape seds. Variety CT EC ECG EGCG B1 B2 GA Cabernet Sauvignon a 215 F 89.3 F 27.9 F 6.5 F 14.8 F 11.3 F 2.8 F Grenache Rouge a 203 F 86.8 F 18.6 F 9.5 F 10.6 F 6.1 F 3.4 F Merlot a 183 F 83.4 F 58 F 13.5 F 13.5 F 17.6 F 2.7 F andilaria a 454 F 249 F 64.4 F 15.6 F 102 F 69.2 F 10.5 F Agiorgitiko a 245 F 172 F 41.3 F 10.9 F 31.9 F 36.1 F 17.9 F Xinomavro a 36.7 F 17.5 F 0.1 F 0.1 F 0 F 0.1 F 0.7 F Limnio a 51.3 F 20.1 F 13.8 F 0.3 F 0 F 0.1 F 1.2 F Turkish Varieties b 471-2580 D 249-1688 D 32-1150 D 79-255 D 56-194 D 41-160 D NA Sp?tburgunder c 376 D 612 D 92 D NA 499 D 298 D NA Samtrot c 464 D 331 D 198 D NA 207 D 273 D NA M?ller-Thurgau c 217 D 206 D 80 D NA 121 D 121 D NA Kerner 88 D 223 D 41 D NA 61 D 116 D NA Schwarzriesling c 238 D 266 D 169 D NA 91 D 122 D NA White grape d 12-50 D 11-31 D 1.3-6.7 D NA 20-62 D 1.9-3.3 D NA Red grape d 8.2-27 D 6-21 D 3.2-7 D NA 7.4-17 D 2.1-4.1 D 0.7-1.0 D Vinifera e 25-244 D 24-193 D NA NA 11-62 D 29-93 D NA Hybrid e 21-155 A 23-284 A NA NA 3-60 A 9-106 A NA Labruska e 37-58 A 55-97 A NA NA 7-11 A 29-75 A NA Weiser Riesling f 79 D 67.5 D 45.8 D NA 105.4 D 50.6 D NA References: a - Guendez et al. 2005; b - Bozan et al. 2008; c - Maier et al. 2009; d - Montealegre et al. 2006; e - Fuleki and Ricardo da Silva 1997; f - Kamerer et al. 2005. Al quantities expresed in mg/100 g sed. CT: (+)-catechin; EC: (-)-epicatechin; ECG: (-)-epicatechin galate; EGCG: (-)- epigalocatechin galate; B1: proanthocyanidin B1; B2: proanthocyanidin B2; GA: galic acid; D : dry weight; F : fresh weight; A : air dried; NA: not analyzed. 27 Of the varieties reported on a dry weight basis, the highest concentration of catechin was found in the seds of Okuzgozu variety at 2580 mg/100 g, and the highest epicatechin concentration was found to be 1688 mg/100 g dry mass in Senso seds (Bozan et al., 2008). Okuzgozu and Kalecik Karasi grape seds had the highest epicatechin galate (1150 mg/100 g) and epigalocatechin galate (255 mg/100 g) contents, respectively (Bozan et al., 2008), while the highest proanthocyanidin B1 and B2 content was observed in Sp?tburgunder grape seds measuring up to 499 mg/100 g and 298 mg/100 g, respectively (Maier et al., 2009). Of the varieties measured on a fresh weight basis, the most remarkable one was the seds of Mandilaria variety that had the highest levels of catechin at 454 mg/100 g, epicatechin at 249 mg/100g, epicatechin galate at 64.4 mg/100 g, epigalocatechin galate at 15.6 mg/100 g, procyanidin B1 at 102 mg/100 g, and procyanidin B2 at 69.2 mg/100 g, along with the greatest total polyphenolic concentration and the second highest concentration of galic acid at 10.5 mg/100 g (Guendez et al., 2005). Catechin and epicatechin were, on average, the most abundant polyphenolic compounds in the seds of the analyzed grape varieties. Of the rest of the compounds analyzed, proanthocyanidin B2 and epicatechin galate were the next most abundant, depending on the variety of grape and method of estimation. Grape parts also may difer in the concentrations of individual phenolic compounds (Monagas et al., 2003; Kamerer et al., 2004; Yilmaz & Toledo, 2004; Montealegre et al., 2006; Iacopini et al., 2008; Huang et al., 2009). Table 2.4 compares the levels of catechin, epicatechin, and procyanidin B1 in the seds and skins of eleven grape varieties, including four red and six white. 28 Table 2.4 Polyphenolic composition of seds and skins of red and white grape varieties. Catechin Epicatechin Procyanidin B1 Variety Seed Skin Seed Skin Seed Skin Weiser Riesling 2002 a 79.0 D 22.7 D 67.5 D 13.5 D 105.4 D 19.2 D Riesling b 40.0 F 1.4 F 16.0 F trace 62.0 F 1.2 F Merlot b 24.0 F 2.5 F 21.0 F 1.3 F 17.0 F 2.1 F Cabernet Sauvignon b 27.0 F 1.7 F 13.0 F 0.6 F 15.0 F 1.2 F Chardonnay b 39.0 F 2.3 F 31.0 F 0.6 F 38.0 F 2.3 F Sauvignon Blanc b 20.0 F 1.0 F 13.0 F 0.3 F 25.0 F 1.6 F Moscatel b 35.0 F 1.6 F 12.0 F 0.3 F 33.0 F 2.1 F Gew?rztraminer b 50.0 F 1.9 F 15.0 F 0.8 F 46.0 F 4.8 F Viogner b 12.0 F trace 11.0 F trace 20.0 F trace Cencibel b 8.2 F 2.2 F 6.0 F 0.8 F 7.4 F 2.2 F Shiraz b 12.0 F 0.9 F 13.0 F 0.7 F 10.0 F 0.8 F References: a - Montealegre et al. 2006; b - Kamerer et al. 2005. Al quantities expresed in mg/100 g sed. D : dry weight; F : fresh weight. 29 The tested seds exhibited higher levels of catechin, epicatechin, and procyanidin B1 than their corresponding skin samples on a per sample weight basis regardles of grape skin color (Table 2.4). This observation is supported by the results from a number of other studies (Monagas et al., 2003; Yilmaz & Toledo, 2004). In 2003, Monagas and colleagues examined the levels of anthocyanins and flavanols in the skin, seds, and wine of Tempranilo, Graciano, and Cabernet Sauvignon grapes. Phenolic compositions difered greatly betwen the skin and seds of same grapes and betwen seds from diferent grape samples (Monagas et al., 2003). The seds had higher levels of all detected flavanol and anthocyanin compounds than the corresponding skin samples. For instance, Tempranilo seds contained (-)-epicatechin at a level of 0.62 mg/g, whereas the skin had a (-)-epicatechin concentration of 0.079 mg/g on a dry weight basis (Monagas et al., 2003). Another study by Yilmaz and Toledo also detected higher levels of catechin in the seds as opposed to the skins of Chardonnay and Merlot grapes (Yilmaz & Toledo, 2004). It was also reported from this study that seds of both varieties had greater galic acid concentrations (15 and 10 mg/100g dry seds compared to 5 and 3 mg/100 g dry skin, respectively) for Chardonnay and Merlot grapes. It needs to be pointed out that a few studies reported the levels of quercetin, resveratrol, rutin, myricetin, cyanidin glucoside and other phenolic compounds in grape skins, but did not report their presence in the seds (Pastrana- Bonila et al., 2003; Iacopini et al., 2008; Huang et al., 2009). This indicated that grape seds may have unique phenolic composition compared to the skin parts and could be utilized for diferent beneficial effects. 30 Trans-Resveratrol Trans-Resveratrol (trans-3,5,4?-trihydroxystilbene, sometimes referred to as resveratrol) is a polyphenolic compound naturaly present in grape skins and seds (Fig. 2.1C). Trans- resveratrol has been shown to have a number of health beneficial effects including inhibition of platelet aggregation, anti-inflamatory activity, antioxidant properties, capacity to reduce the risk of cancer and cardiovascular disease, and possible longevity promoting effect (Howitz et al., 2003; Wood et al., 2004; Yilmaz & Toledo, 2004; Li et al., 2006; Iacopini et al., 2008). The levels of trans-resveratrol in grape seds and skin were investigated (Li et al., 2006; Iacopini et al., 2008). Extractable or available amounts of trans-resveratrol in the seds and skin of 120 grape varieties grown in two years have been compared (Li et al., 2006). Methanol at a solvent- solid ratio of 5 mL for each gram of frozen seds was used for trans-resveratrol extraction at 25?C for 48 hours in dark. Results from this study, measured using HPLC-UV, showed that grape seds contained high levels of trans-resveratrol (Li et al., 2006). This study also showed that both variety type and growing conditions altered its level in seds and skin. The level of trans-resveratrol varied from 1.06 to 17.03 !g/g in the tested grape seds, and varied from 0.56 to 145.11 !g/g skin on per fresh weight (Li et al., 2006). Importantly, seds of some varieties of grape contained greater level of trans-resveratrol than their counterpart skin samples. Taken together, these data indicated that grape seds and skin may serve as dietary sources for trans- resveratrol. In contrast, a recent study detected no trans-resveratrol in the grape seds, while significant level of trans-resveratrol was found in grape skin samples under the same experimental conditions (Iacopini et al., 2008). Ethanol:water:hydrochloric acid (0.12 M) at 70:29:1 (v/v/v) was used for extracting resveratrol and the extraction was performed in 4 hours. 31 While the diference in grape varieties could be a possible explanation for the absence of extracted trans-resveratrol from grape seds, it could also have been partialy due to a diferent extraction method. Antioxidant Properties Antioxidative Properties of Grape Seeds Grape seds have been shown to have antioxidative properties (Jayaprakasha et al., 2003; Janisch et al., 2006; Kim et al., 2006; Yilmaz & Toledo, 2006; Bozan et al., 2008; Iacopini et al., 2008; Poudel et al., 2008). The seds of V. vinifera variety Bangalore blue grapes grown in India were analyzed for their capacity to reduce Mo (VI) to Mo (V) and to quench 2,2-diphenyl-1- picrylhydrazyl (DPPH) radicals (Jayaprakasha et al., 2003). The seed extracts exhibited dose- dependent DPPH ? scavenging property and reducing power under the experimental conditions. In 2008, defated seds of eleven grape varieties grown in Turkey were extracted with acetone:water(70:30, v/v) containing 0.5% acetic acid at 50?C and the extracts were evaluated for their free radical scavenging activities (Bozan et al., 2008). All eleven seed extracts showed significant ability to directly react with and quench peroxyl (ORAC) and DPPH radicals (Bozan et al., 2008).The greatest ORAC value was 3.0 mol trolox equivalents (TE)/g dry seds determined in the Okuzgozu grape seds, and the lowest ORAC value was 1.4 mol TE/g for Bogazkere seds; a 2-fold diference (Fig. 2.2). 32 Figure 2.2 Oxygen radical absorbance capacity (ORAC) of grape seed extracts. Adapted from Bozan et al. (2008). Results were reported as millimoles of trolox equivalents per gram (mmol TE/g) The DPPH radical scavenging capacity was reported using EC 50 values, which is the required antioxidant concentration to quench 50% of the radicals in the system under the assay conditions. The Papaz Karasi grape seds had the smallest EC 50 value against DPPH radicals, which represented the greatest DPPH radical scavenging capacity, whereas the Okuzgozu seds had an EC 50 value of 2.89 ?g/mL, the third strongest DPPH radical scavenging ability among the eleven grape seds (Bozan et al., 2008). DPPH radical scavenging capacity was also determined for five wild grapes in another study (Poudel et al., 2008). Another study showed the DPPH ? and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) radical (ABTS ? + ) scavenging capacities of 12 varieties of V. vinifera grape grown in Turkey (Yemis et al., 2008). All tested grape seds 33 showed significant scavenging capacity against both DPPH ? and ABTS ? + . While the diferent grape varieties ranked diferently in ABTS ? + scavenging and DPPH ? scavenging, both DPPH ? and ABTS ? + scavenging capacities were significantly correlated to the total phenolic contents of the seds (Yemis et al., 2008). In 2009, a study compared grape seed extract with ascorbic acid and chlorhexidine for their ability to directly react with and quench chemicaly generated ABTS cation radicals (Furiga et al., 2009). The grape seed extract had the greatest ABTS ? + scavenging capacity under the experimental conditions. These results indicated that these grape seds may difer in the content and compositions of their antioxidative components, which might have interacted with peroxyl (ORAC), ABTS cation, and DPPH radicals in diferent manners under the assay conditions. It is also widely acepted that individual antioxidant activity asays difer in their determination principles and antioxidant activity estimation depends on the asays selected. Grape seds have also been compared with other grape parts for antioxidant properties. The pulp, peel, and seds of red rose grape from a Chinese local market were compared for their ferric reducing/antioxidant power (FRAP) using FeSO 4 as the standard (Guo et al., 2003). The seds had a FRAP value of about 56 mol/100 g, which was much greater than 0.49 and 11 mol/100 g for pulp and skin, respectively, on a per fresh weight basis. In 2008, seds and skins of ten native Tuscan and international red grape samples were compared for their DPPH radical scavenging abilities (Iacopini et al., 2008). The IC 50 values, which are the required antioxidant concentration to quench 50% of the radicals in the assay mixtures under the experimental conditions, were estimated (Fig. 2.3). 34 Figure 2.3 Antioxidant activity of grape seed and skin of different grape varieties native to Tuscan. Values were reported as IC 50 values in galic acid equivalents per liter (GAEL), which was the concentration required to inhibit 50% of radicals (Iacopini et al., 2008). The antioxidants were extracted using ethanol:water:hydrochloric acid (0.12 M) (70:29:1, v/v/v). The seds of Sangiovese clone ISV RC1 and the skin of Merlot grapes had the strongest DPPH radical scavenging capacity with the lowest same IC 50 value of 1.74 mg GAE/L. In 2006, Yilmaz and Toledo compared the peroxyl radical scavenging capacity (ORAC) for the seds and skin of Merlot and Chardonnay grapes (Yilmaz & Toledo, 2006). The seds had ORAC values of 345 and 638 ?mol TE/g for Merlot and Chardonnay grapes, respectively, which were much higher than that of 70 and 103 ?mol TE/g for the counterpart skin samples on a per dry weight basis (Yilmaz & Toledo, 2006). 35 Recently, Choi and Lee (2008) reported that a tocotrienol-rich fraction prepared from Campbel Early grape seds had scavenging capacity against ABTS ? + and DPPH ? , Fe 2+ chelating activity, reducing power, and inhibition of linoleic acid oxidation. These results agreed with the observations from an earlier study showing that Chardonnay grape seed flour extract could suppres overal lipid peroxidation and prevent oxidative loss of longer chain !-3 polyunsaturated faty acids in fish oil (Luther et al., 2007). The seed flour was the residue from seed oil preparation by cold-presing. This study also demonstrated peroxyl radical scavenging capacity (ORAC) of Chardonnay seed flours, which was more than 6 times higher than the black raspberry seed flour on a per dry flour weight basis under the same assay conditions. DPPH ? scavenging activity of the grape seed extract at a final concentration of 26 mg seed flour equivalent/mL was similar to that of the black raspberry seed extract, and greater than that observed for 50 ppm of the mixed tocopherol (Luther et al., 2007). In addition, the cold-presed Chardonnay and Pinot Noir grape seed flours were evaluated for their ORAC, DPPH ? scavenging, and Fe 2+ chelating capacities (Parry et al., 2006). Individual grape phenolic compounds have been investigated for their antioxidant properties in human low-density lipoprotein (LDL) (Janisch et al., 2006). A reduced degree of LDL oxidation has been asociated with a lower plaque formation in arteries (Stocker & Keaney, 2004). The required concentration for galic acid, catechin, epicatechin and procyanidins B1, B2, B3, C1 and EB5 to achieve the maximum lag time measured as diene formation was determined in the study. Procyanidin EB5 was the most efective compound to prevent lipid peroxidation in the LDL under the experimental conditions (Fig. 2.4). 36 Figure 2. 4 Inhibitory effects of individual grape phenolic compounds on lipid peroxidation in human LDL. Results were expresed in nanomolar of each phenolic compound required to reach maximum lag time (adapted from Janisch et al., 2006). The lag time was determined by photometricaly tracking acumulation of conjugated diene formation through absorbance at ! = 234 nm. 37 The ability of these compounds to quench hydroxyl and peroxyl anion radicals generaly followed the order of suppresing LDL oxidation (Janisch et al., 2006). Earlier in 1991, catechin, epicatechin, epicatechin galate, procyanidins B2 and B5 and C1, procyanidin B2 galate, as well as procyanidin trimer 2 and trimer 3 showed peroxide anion radical scavenging capacity at pH 7.5 and pH 9.0 conditions (da Silva et al., 1991). In 2008, individual grape phenolic compounds such as catechin, epicatechin, rutin, trans- resveratrol, and quercetin were shown to have scavenging capacity against DPPH ? and peroxynitrite (Iacopini et al., 2008). In addition, galic acid and catechin were reported for their scavenging capacity against DPPH ? , peroxide anion (O 2 ? - ) and hydroxyl (HO ? ) radicals, and their reducing power (Spranger et al., 2008). Finaly, trans-resveratrol was compared with other grape phenolic compounds for antioxidant properties. Trans-resveratrol exhibited a peroxyl radical scavenging capacity (ORAC value) of 29.06 !mol trolox equivalent (TE)/mg, while catechin had an ORAC value of 20.53 ?mol/mg under the same assay conditions, which was followed by epicatechin, galocatechin, galic acid, and elagic acid with a range of ORAC value from 20.53 to 3.88 !mol TE/mg (Yilmaz & Toledo, 2004). However, trans-resveratrol had a weaker DPPH radical scavenging capacity than quercetin, catechin, epicatechin, and rutin acording to their IC 50 values against DPPH radicals (Iacopini et al., 2008). It needs to be pointed out that grape phenolic antioxidants have shown other beneficial effects besides their antioxidative properties. These beneficial effects may include but are not limited to anti-proliferative activity against cancer cells (Parry et al., 2006; Choi & Lee, 2008), antibacterial activity (Jayaprakasha et al., 2003; Luther et al., 2007), lifespan extension (Howitz et al., 2003; Wood et al., 2004), and prevention of 38 cataract formation (Yamakoshi et al., 2002). These beneficial properties may not be mediated by their antioxidant activities, but rather by other metabolic pathways. Effects of Post-Harvest Treatments on Grape Seed Antioxidants Effects of Thermal Treatment The bioavailability of bioactive food factors is critical for their beneficial effects. The bioavailability depends on their original concentration in the raw ingredients and changes during post-harvest treatments, such as chemical and biochemical reactions during storage and ingredient procesing, as well as their interactions with other components during food formulation and procesing. Effects of thermal treatment on antioxidant availability in grape seds has been investigated (Kim et al., 2006). Whole and powdered grape seds (V. vinifera, Campbel Early) were heated at 50, 100, 150, or 200 ?C for 10, 20, 30, 40, 60, 90, and 120 minutes. These seed preparations were compared to the control, which are the seds without thermal treatment, for their TPC and antioxidant properties. As shown in Table 2.5, TPC values decreased in both whole and powdered grape seds in a temperature and time dependent matter except the powdered seds kept at 50 ?C, indicating the possible loss of phenolic components due to thermaly induced chemical reactions, such as oxidation. 39 Table 2. 5 Efects of thermal treatment on total phenolic content (mM TAE) of whole and powdered grape (V. vinifera, Campbel early) seds. a Temperature Heating Time (min) (?C) 0 10 20 30 40 60 90 120 WGSE 50 0.380 0.317 0.260 0.303 0.300 0.313 0.442 0.330 100 0.380 0.326 0.347 0.414 0.407 0.520 0.458 0.390 150 0.380 0.392 0.348 0.444 0.575 0.484 0.358 0.319 200 0.380 0.254 0.189 0.163 0.115 0.179 0.179 0.113 PGSE 50 0.380 0.344 0.332 0.451 0.424 0.444 0.400 0.515 100 0.380 0.555 0.296 0.359 0.375 0.378 0.418 0.185 150 0.380 0.340 0.417 0.427 0.483 0.407 0.319 0.196 200 0.380 0.190 0.185 0.269 0.160 0.151 0.067 0.064 a Referenced from Kim et al. 2006 TAE: tannic acid equivalents; WGSE: whole grape sed extract; PGSE: powdered grape sed extract. 40 It should also be noted that powdered grape seds demonstrated a higher TPC value than the whole seed counterparts at all time points when kept at 50 and at 100 ?C for 10 min, but the whole seds had a greater TPC value than the powdered seed counterparts when they were kept at 150 and 200 ?C for 10-120 min, or at 100 ?C for 20- 120 min. These results suggest that mild thermal treatment, such as heating at 50 ?C for a short time period, may enhance the extractable or available level of phenolics in the powdered grape seds. This also suggests that the seed matrix may protect phenolic compounds during thermal treatment. This observation may be explained by the overal effects of thermal cleavage of asociations betwen phenolics and the seed matrix, and the increased surface area of the powdered seds (Meyer et al., 1997; Cheng et al., 2006; Kim et al., 2006). Heat treatment has been shown to possibly increase release of phenolic compounds as it may convert insoluble bound phenolic compounds into soluble phenolic compounds (Kim et al., 2006; Moore et al., 2009). Thermal treatment also altered the antioxidant property of grape seds (Kim et al., 2006). As shown in Fig. 2.5, increase of the extractable DPPH radical scavenging capacity was observed in whole and powdered grape seds kept at 50 and 100 ?C for 60 and 120 min, while heating at 200 ?C for 60 and 120 min decreased DPPH radical scavenging activity in whole and ground seds (Kim et al., 2006). Kim and colleagues (2006) also reported the alteration of reducing power of grape seds by thermal treatment. 41 Figure 2. 5 Effects of thermal treatment and particle size on antioxidant stability in whole and powdered grape seeds (adapted from Kim et al., 2006). The radical scavenging activity was estimated by the formula: % DPPH radical scavenging activity = (1- sample absorbance/control absorbance) ! 100%. 42 Table 2. 6 Efects of diferent extraction times and crushing on total phenolic, anthocyanin, and flavanol contents of grape sed extracts. a Extraction Time Total Phenolics (mg GAE/L) Anthocyanins (mg ME/L) Flavanols (mg CCE/L) Cab. Sauvignon (whole seds) 1 min 565 718.0 0.0 1 h 686 696.0 0.0 4 h 771 793.8 0.0 24 h 737 705.8 8.9 165 h (! 7 days) 890 746.4 0.0 Cab. Sauvignon (crushed seds) 1 min 1780 791.7 133.7 1 h 1868 879.9 122.7 4 h 1930 867.8 140.9 24 h 2015 856.2 156.0 165 h (! 7 days) 2138 775.2 167.4 P. Sirah late (whole seds) 1 min 1115 1708.2 5.9 1 h 1136 1483.6 0.0 4 h 1163 1558.9 5.5 24 h 1183 1477.3 21.5 165 h (! 7 days) 1367 1564.7 99.4 P. Sirah late (crushed seds) 1 min 1741 1337.2 93.4 1 h 1820 1644.2 101.8 4 h 1966 1463.4 137.6 24 h 1964 1513.2 173.6 165 h (! 7 days) 2094 1344.0 168.0 a Data from Meyer et al. 1997 GAE: galic acid equivalents; ME: malvin equivalents; CCE: catechin equivalents. 43 Effects of Particle Size Particle size of the botanical materials including food and nutraceutical ingredients may affect the stability of their important components during storage and post-harvest treatments such as ingredient and food procesing procedures (Cheng et al., 2006). In 2006, Kim and others theorized that reduction of particle size, through the grinding of grape seds, might contribute to the increased available amount of total phenolics kept at 50 ?C (se Effect of Thermal Treatment section). In addition to this observation, Meyer and colleagues reported that crushed seds might have higher levels of extractable total phenolics, benzoic acids, flavanols, and cinnamates, but not anthocyanins, than their whole seds counterparts (1997). Under the same analytical conditions, crushed Cabernet Sauvignon grape seds had a TPC value of 1780 mg GAE/L and the TPC was 565 mg GAE/L for the whole seds (Table 2.6). Effects of Storage Conditions Storage conditions have been found to affect antioxidant properties in grapes and grape seds (Hatzidimitriou et al., 2007; Romero et al., 2008). Hatzidimitriou and colleagues analyzed the effects of storage at three diferent relative humidity (RH) levels, 33, 53, and 75%, on TPC of grape seed extracts (2007). TPC decreased from 438 to 327 and 438 to 344 mg GAE/g dry extraction respectively for the grape seds kept at 33 and 53% RH in 50 days, and dropped from 438 to 234 mg GAE/g for seds stored at 75% RH (Hatzidimitriou et al., 2007). DPPH ? scavenging abilities of the grape seed extracts were slightly reduced at all tested RH conditions. Interestingly, storage at either higher RH level 44 or for longer time could enhance the galic acid level in the grape seds, but would reduce both catechin and epicatechin contents (Hatzidimitriou et al., 2007). Considerations in Antioxidant Property Estimation for Grape Seeds Many factors may alter the overal estimation of antioxidant properties of grape seds. It is widely acepted that mistakes made during sample preparation cannot be corrected in the later analytical steps. In 2005, Pinelo and others investigated the effects of solvent, temperature, and solvent-solid ratio on estimation of total phenolic content and radical scavenging capacity of grape pomace, stem, seds, and skin. The extraction temperature and solvent-solid ratio were critical in the extraction eficiency of phenolic antioxidants (Pinelo et al., 2005). It was also noted that methanol was most efective for phenolic extraction, while ethanol extracted the highest level of soluble material under the experimental conditions, suggesting the importance of solvent type in antioxidant property estimation. This conclusion was supported by findings from a recent study (Yilmaz & Toledo, 2006). Results from this study showed that methanol, ethanol, and acetone with diferent levels of water difered in their capacities in extracting phenolic components from grape seds and skin (Yilmaz & Toledo, 2006). The critical role of temperature and solvent-solid ratio on phenolic extraction from grape seds was confirmed by a kinetic study performed by Buci!-Koji! and colleagues (2007). In addition, pH and particle size might alter the extraction eficiency of phenolic antioxidants from grape seds (Janisch et al., 2006; Makris et al., 2007; Buci!-Koji! et al., 2007). 45 Summary Grape seds and seed fractions may serve as dietary source of natural phenolic antioxidants, such as flavanols, anthocyanins, and simple phenolic acids. Genotype, growing conditions, and post-harvest treatments may alter the content of antioxidants in grape seds. This may be a chalenge in developing grape seds-based nutraceutical ingredients for human utilization. Multi-mechanisms such as radical scavenging and chelating reactions may be involved in the antioxidative actions of these phenolic compounds. Additional research is required to investigate their health beneficial effects and possible side effects to promote their application in improving human health. Incorporation Into Functional Foods Functional Foods: An Overview The study of functional food is a popular and constantly evolving area of food science. The term functional food has been defined by many diferent organizations worldwide. There is no universaly acepted definition of this term, but the International Food Information Council (IFIC) and the Institute of Food Technologists (IFT) define functional foods as a food or food component that has health beneficial qualities beyond basic nutrition (International Food Information Council, 2009). More specificaly, an IFT report extended the definition by stating that functional foods ?provide esential nutrients often beyond quantities necesary for normal maintenance, growth, and development, and/or other biologicaly active components that impart health benefits or desirable physiological effects? (Institute of Food Technologists, 2009). The American Dietetic Asociation (ADA), on the other hand, believes all foods are functional to a certain extent. 46 In this definition, functional foods are those ?that include whole foods and fortified, enriched, or enhanced foods have a potentialy beneficial effect on health when consumed as a part of a varied diet on a regular basis at efective levels? (American Dietetic Asociation, 2009). Political bodies, however, have been slow to resolve the controversy of defining functional foods. The United States Food and Drug Administration (FDA) offers no definition regarding functional foods. There is also no regulatory framework for foods that are marketed as functional food in the United States, either. In fact, Japan is the only country in the world that formaly regulates functional foods as a distinct category (American Dietetic Asociation, 2009). The phrase ?functional food? was first used in the 1980s in Japan to refer to food fortified with special constituents that had positive physiological effects (Kwak & Jukes, 2001). As functional foods have become more and more popular in recent years, there has been increasing presure on the FDA to define functional food more clearly and regulate this $20.5 bilion market (Burdock et al., 2006). A similar situation exists in the European Union (EU). Under EU Food Law, no framework for functional food regulation is available. Thus, the recent years have seen calls for more definition and regulation of functional food in Europe, as well (Coppens et al., 2005). Antioxidants in Functional Foods Many nutrients have been added to functional foods with the purpose of improving their health beneficial properties. Antioxidants, specificaly, have been succesfully incorporated in a multitude of functional food products. Tortilas, for example, have been 47 developed into a functional food, through the addition of antioxidant-containing bean flours. In an experiment conducted by Anton and collegues, flours were prepared from four varieties of bean (red, navy, pinto, and black) that were subsequently made into tortilas. In comparison to a wheat flour control, the bean flour tortilas were found to have higher levels of protein, total phenolic content (TPC), DPPH radical scavenging capacity, and ABTS cation radical scavenging capacity (Anton et al., 2008). Nutrients added to functional foods, however, are not always succesful in increasing the overal intended health-beneficial properties of the product. An example of this can be found in a study of two foods, pan bread and sugar-snap cookies, which were supplemented with the food additives red palm olein (RPOL) and red palm shortening (RPS), with the intention of increasing the vitamin E content of these food products (Al-Saqer et al., 2004). The results of this experiment showed that the total tocopherol content and the total tocotrienol content of the breads (whole-wheat, brown, and white) were substantialy lowered in those containing 75% RPOL (192 and 407 mg/kg fat, respectively) or RPS (101.4 and 300 mg/kg fat, respectively), when compared to the bread samples made solely with control shortening that had a TPC of 325.0 mg/kg fat and a total tocotrienol content of 468 mg/kg fat. Similarly, total vitamin E content of the cookies containing 100% RPS (367.3 mg/kg fat) and 100% RPOL (489.9 mg/kg fat) was significantly lower than that of the bread containing 100% control shortening (781.6 mg/kg fat) (Al-Saqer et al., 2004). A number of factors influence the efectivenes of antioxidant properties in functional foods containing added antioxidants. A study, for example, conducted by Moore and colleagues (2009), analyzed the effect of specific procesing conditions on the antioxidant properties of a whole-wheat piza crust functional food model. The research looked into the 48 effects of these conditions on ORAC, hydroxyl radical scavenging capacity (HOSC), DPPH radical scavenging capacity, ABTS radical scavenging, TPC, and ferulic acid content, of whole-wheat piza crust. The factors that were studied included bran particle size, dough fermentation time, and baking time/temperature. Results showed that bran particle size had no substantial effect on antioxidant properties. Similarly, little diference was noted when dough fermentation time increased (except HOSC which increased a maximum of 28%). It was noted that as baking temperature increased, from 204 ?C to 288 ?C with a 7 min bake time, all antioxidant properties increased up to 82%. An increase in bake time from 7 to 14 min at 204 ?C increased some of the antioxidant properties. These results show that the functional food model (whole-wheat piza crust) may have an increased antioxidant availability as dough fermentation time, baking time, and baking temperature are increased (Moore et al., 2009). Bread as a Functional Food Model Bread has been shown to be an efective model for a variety of functional foods including: antioxidants, faty acids, anti-phytases, gluten, !-aminobutyric acid (GABA), and fiber. Ingredients of bread can be substituted with little negative effect on rheological properties or sensory quality (Saiz et al., 2007). Flour has been substituted with chickpea, amaranth, quinoa, buckwheat, grape sed, flaxsed, barley, rice bran, wheat bran, sugarcane bagase, carob fiber, inulin, and pea fiber (Gill et al., 2002; Sangnark & Noomhorm, 2004; Penela et al., 2008; Conforti & Cachaper, 2009; Hu et al., 2009; Lin et al., 2009; Coda et al., 2010; Peng et al., 2010). The novel bread products all were considered aceptable by sensory panels, indicating the possibility of commercial applications of bread as a functional food. 49 Additives in Bread Models Antioxidants Increased levels of antioxidants in bread were achieved with addition of diferent ingredients as well as novel procesing methods. Addition of a chickpea, amaranth, quinoa, and buckwheat flour blend to a bread product, resulted in increased level of phenolics (Coda et al., 2010). The non-conventional bread product also underwent sourdough fermentation and exhibited the highest levels of phenolics, followed by non- conventional bread baked under normal conditions, wheat bread exhibited the lowest levels. Non-conventional bread baked under both conditions also exhibited high DPPH- radical scavenging ability. Sourdough fermentation produces !-aminobutyric acid (GABA), which functions as an anti-hypertensive, diuretic, tranquilizer, and prevents diabetes (Coda et al., 2010). Wheat flour was substituted with unhusked and husked buckwheat. Husked buckwheat bread contained more insoluble "-glucan, which provides an imunostimulating effect. Sensory analysis, using the 7 point hedonic scale where 1, 4, and 7 respectively representing strongly dislike, neither like nor dislike, and strongly like, showed that all three breads (husked, unhusked buckwheat, and wheat breads) were aceptable to consumers, and both husked and unhusked buckwheat breads scored higher in flavor and mouth-feel than the wheat bread. The flavanoids rutin and quercetin were found in substantial quantities in husked buckwheat bread, and in smaller quantities in unhusked buckwheat and wheat bread products. The buckwheat-enhanced bread also showed enhanced antioxidant properties, especialy unhusked buckwheat bread product (Lin et al., 2009). 50 Addition of grape seed extract to white bread resulted in increased antioxidant activity and decreased levels of the detrimental glycation end-product in bread, N!- (carboxymethyl) lysine (CML). CML is considered a toxoid in food and a biomarker asociated with oxidative stres, artherosclerosis and diabetes. The grape seed bread showed little change in the sensory analysis. The baking proces reduced antioxidant activity in the bread by 30 to 40 % (Peng et al., 2010). Fatty Acids Quinoa and flaxsed (linsed) bread were prepared and compared for physiochemical properties and underwent a sensory analysis. The flaxsed bread was higher in both lipid level and caloric value. Both breads were low in trans-faty acids, while flaxsed bread exhibited higher levels of saturated, monounsaturated, polyunsaturated, omega-6 and omega-3 faty acids, as well as a lower omega-6 to omega-3 ratio. However, quinoa bread was found to be low in saturated faty acids. Additionaly, consumers preferred quinoa bread to flaxsed bread. During the baking proces, faty acids in both flaxsed and quinoa were lost from the original ingredients (Caldereli et al., 2010). Fiber While the clasification of fiber as a functional food is contested, a significant amount of research has been done on the subject, especialy with bread as the model. Substitution of high-fiber grains can increase fiber content in bread. Barley contains high levels of soluble (digestible) fiber, with higher fiber content in waxy barley flour as 51 opposed to regular barley flour. However, substitution of wheat with barley flour decreased loaf volume and altered the color, firmnes, and texture of the loaves. The addition of barley yielded poor quality bread products. Changes were dependent on barley variety (Gil et al., 2002). Wheat bread has also been supplemented with carob fiber, inulin and pea fiber as sources of fiber. Carob and pea fiber led to softnes in the bread crumb, and the breads supplemented with these two fibers were judged as aceptable. All three sources of fiber could be used to increase dietary intake. The level of soluble dietary fiber was greatly increased with inulin-added bread. The fiber with the greatest potential for increasing fiber intake, balanced with consumer aceptability was carob fiber (Wang et al., 2002). Additionaly, diferent procesing methods can lead to more nutritious functional food products. Extrusion cooking alows for improvements in nutritional quality of dietary fiber. Extrusion is most commonly used to produce breakfast cereals, and involves heating food under a high degree of presure, then pushing foodstuffs through a series of pores. The use of extruded flour produced more favorable bread than using cooked flour (Gil et al., 2002). Izydorczyk and others found that decreased particle size increased the percentage of water soluble beta-glucans and arabinoxylans, increased starch damage, and increased the level of free phenolics in fiber-enriched breads (2008). Addition of fiber to bread products may have negative effects on the nutritive and sensory properties; however, other compounds can be added to minimize these negative effects. Phytic acid is a compound commonly found in plant seds and grains that forms insoluble complexes with cations, decreasing the bioavailability of nutrients like calcium, magnesium, and potasium. The combination of adding bran and phytate-degrading 52 enzymes alows for the increased fiber from bran without the negative effects of the increased amounts of phytate from the bran (Penela et al., 2008). Sangnark and Noomhorm (2004) found that combining sucrose ester and fiber alowed addition of fiber without the great losses in consumer aceptability. Sugarcane bagase was used for addition of dietary fiber, and sucrose ester was added as an emulsifier to increase the proportion of dietary fiber in white pan bread. Addition of fiber decreased loaf volume, increased firmnes, and negatively affected crumb color and bread texture, while addition of sucrose positively influenced bread properties. Diet and Health Effects Supplementation With rising health care costs and research indicating that diet is directly correlated with the incidence of disease, Americans have focused more atention on consuming their daily nutrients through functional foods (Milner, 2000). However, supplementation along with functional foods can provide a means for Americans to get the health benefits of these nutrients and antioxidants within their diet. The major determinants of the aging proces are environmental and lifestyle factors, particularly diet. With aging, the likelihood of disease is greater, and high levels of oxidative stres and free radicals can cause many chronic ilneses, including arthritis, cardiovascular disease, and cancer (Meydani, 2000). Recent research indicates that certain nutritional supplements can counteract or minimize the effects of these diseases. Many vitamins, including vitamin E, have been found to have antioxidative properties. Research has shown that vitamin E is often supplemented with functional foods, and when 53 consumed it can have positive cardiovascular and imune effects in the elderly. In one study conducted, healthy subjects over the age of 60 who consumed 800 IU of vitamin E over a one-month period were found to have reduced production levels of prostaglandin ES (PGE2), a lipid based mediator molecule, and plasma lipid peroxide concentrations (Meydani, 2000). The vitamin E supplementation was also shown to enhance cell- mediated imunity. The preventive effects of vitamin supplements on age-related diseases were also observed in a study conducted by Zandi and colleagues (2004). In the study, researchers examined the relationship betwen vitamins C, E, and B-complex supplements and Alzheimer?s disease. The study was designed as a cross-sectional study of 4740 elderly respondents, with notes made of their dietary supplemental use over a 5-year period. The results showed that vitamin E used in combination with vitamin C exhibited the greatest protective effects against Alzheimer?s disease. These results support previous study findings that show that vitamin supplementation plays a key role in reducing the occurrence of age-related diseases as a result of its antioxidative properties. Antioxidants can also be supplemented into diets, and reduce oxidative stres in the body. For example, in one study, patients with Type-II diabetes were given a 200-mg !-tocopherol supplement over a two-month period. After the two-month period, the antioxidant supplementation was found to have decreased blood-lipid peroxide concentrations without afecting antioxidative activities of the patients (Park & Choi, 2002). In other studies, antioxidants were supplemented into whole foods and their health benefits were studied (Vatem et al., 2005). Fruits and vegetables have been found to 54 decrease the incidence of disease, which has increased the desire to supplement diets with the phenolic compounds that these foods contain. There is also evidence that suggests that phenolic phytochemicals in whole foods are more beneficial to human health than individual phenolic phytochemicals alone because of diferences in bioavailability. As a result, more research has been conducted on how phenolic compounds can be added into a wider range of foods. For example, cranberries are a natural source of phenolic compounds with antioxidants that have been shown to have positive efects against urinary and cardiovascular diseases (Yan et al., 2002). By contributing their phenolic compounds, cranberries might be used to enrich other functional foods and promote health benefits (Vatem et al., 2005). Another instance in which antioxidants were found to supplement diets can be seen in tea extracts. Researchers sought to determine whether green tea polyphenols could exert LDL-resistant effects in the body. LDL can react with free radicals and become oxidized. In its oxidized state, LDL can damage tisue and underlying smooth-muscle cells, and inflame arteries, which can result in atherosclerosis (Russel, 1999). In a study by Miura and others (2000), male subjects were all asked to follow a prescribed dietary regimen for two weks. After the two week period, they were divided into a control and experimental group, with the experimental group consuming 300 mg of green tea polyphenol extract twice a day for a wek. After the trial, it was found that supplementation with green tea polyphenols significantly increased LDL resistance to in vivo oxidation, and prevented antioxidative vitamins from being depleted. The polyphenols were found to be useful in decreasing the risk of cardiovascular disease (Miura et al., 2000). 55 In a study conducted in Finland, researchers determined the relationship betwen flavanoid intake and the incidence of lung cancer (Knekt et al., 1997). The cross-sectional study involved 9959 men and women from the ages of 15 to 99. After 24 years, researchers found that the mean flavanoid intake was 4.0 mg per day with intake amounts ranging from 0 to 41.4 mg. Those subjects who consumed the highest level of flavanoids in their diet, which was greater than 4.8 mg/day of flavanoid for men and greater than 5.5 mg/day flavanoid for women, reduced their risk for lung cancer by 50%. Specificaly, those subjects who consumed apples as part of their diet had a decreased risk for lung cancer. Overal, it was found that there was an inverse relationship betwen flavanoid consumption and the risk for lung cancer (Knekt et al., 1997). Other studies have investigated the health benefits of diets enriched with antioxidants. In a study conducted by Rebrin and others (2005), the effects of two diferent dietary mixtures, one consisting of vitamin C, vitamin E, L-carnitine, and lipic acid and another consisting of vitamin C, vitamin E, and micronutrients with bioflavanoids, polyphenols, and carotenoids, were studied. The first dietary mixture was fed to a group of mice for 8 months, and the second dietary mixture was fed to another group of mice for 10 months. Both diets were found to have reductive effects in plasma (Rebrin et al., 2005). The first dietary mixture was found to have little effect on the glutathione (GSH) redox status in tisue homogenates or mitochondria. However, for the second dietary mixture, gender and tisue specific effects on the glutathione redox were observed. For example, there was an increase in serum cysteine concentrations in female mice, while GSH elevation occurred only in the homogenates of male kidney and skeletal muscles. 56 Functional Foods Like dietary supplements, functional foods have been shown to have health benefits. The components of functional foods may help to reduce the risk of disease by cooperative and synergistic action (Shahidi, 2004). However, some antioxidants, such as !-carotene, have been found to have no effect or even be detrimental in supplement form because of a complex synergistic nature (Omenn et al., 1996). Consequently, functional foods may provide a physiologicaly advantageous combination of antioxidants. Several types of functional foods have been developed to fulfil a variety of needs. These foods are also varied in the matrix used to aceptably deliver the as target nutrients. By incorporating health beneficial compounds, commonly consumed food may be modified into healthier products. Some of these functional foods have been found to have significant physiological effects. In one study, biscuits were enriched with vitamin B12, folic acid, vitamin C, and prebiotic fiber (Boobier et al., 2007). Subjects consumed four biscuits per day in conjunction with their normal diet. After 28 days of eating the biscuits, the levels of homocysteine and glucose in blood plasma, two factors that have been linked to cardiovascular diseases, decreased. Several studies have incorporated antioxidants into diferent food products to achieve health benefits. Though providing similar benefits, products may difer in the aceptability and bioavailability of the target antioxidants. Snack bars enriched with cocoa flavanol were found to lower total and LDL cholesterol levels (Polagruto et al., 2006). The study participants were given 2 servings per day for six weks. Similarly, milk fortified with phytosterols was found to reduce cholesterol levels in healthy and 57 hypercholesterolemic subjects after only 15 days (Goncalves et al., 2007). High blood cholesterol levels can lead to atherosclerosis and an increased risk of thrombosis (Tapiero et al., 2003). Some studies have substituted ingredients with health beneficial properties in place of ingredients that have little to no value added. Fish oil used instead of margarine in bread, was found to significantly increase the quantity of long-chain omega-3 faty acids in blood plasma (Saldeen et al., 1998). A sensory analysis was performed to determine the consumer aceptability of the bread product. Very few people could detect an aroma of fish in the bread baked with fish oil. Functional components were incorporated into bread to yield prebiotic and pre-aox bread (Seidel et al., 2007). The prebiotic bread contained inulin, linsed and soya fibre, while the prebiotic antioxidant bread (pre-aox-bread), also contained green tea powder, herbs and tomato paste. All caloric intake and daily intake of bread was kept the same. The ferric reducing ability of plasma increased after treatment with the antioxidant prebiotic bread in nonsmokers but showed no change in smokers. Carotenoids were increased with the prebiotic antioxidant bread, and ICAM-1 (a stres marker) decreased after consuming the prebiotic bread. This study shows that a bread product, even after the baking proces could potentialy have health-beneficial effects for the consumer. Summary Functional foods, specificaly a bread model, may serve as a dietary delivery source for many health-beneficial compounds. However, diferent additives affect the final product along with variables, such as baking temperatures and time. This may be a 58 chalenge in developing a functional bread model with grape seed-based nutraceutical ingredients. Additional research is required to investigate viability of a functional bread model containing grape seed by-products, the health beneficial effects of such a product, and possible side effects to promote their application in improving human health. 59 Chapter 3: Chemical Composition and Health Properties of the Selected Cold-Presed Grape Seed Flours and Oils Grape (Vitis vinifera) sed flours and oils may function as dietary sources of antioxidants. In this study, chemical tests were performed to quantify the health beneficial factors of Ruby Red, Norton, Chardonnay, Concord, and White grape sed oils and flours. Flours were extracted with three solvents (70% ethanol, 50% acetone, or 70% acetone with 0.5% acetic acid), while oils were extracted with 90% methanol. Extracts were then tested for their total phenolic contents and scavenging ability against peroxyl, hydroxyl and DPH radicals, while oils were tested for carotenoid and tocopherol contents, faty acid composition, and oxidative stability. Lastly, the antiproliferative efects of the flour and oil extracts were tested on HT-29 human colon cancer cels. Based on the resulting data, the cold-presed Chardonnay grape sed flour was identified as a potential functional food ingredient deserving further analysis. Introduction The human body functions through carefully monitored oxidation-reduction reactions, both inside and outside cels, that are used to maintain metabolism and general health. Oxidative stres can shift the delicate balance of oxidation and reduction, causing undesirable elevated levels of free radicals in the body (Hennig et al., 2007). Free radicals are strong oxidative chemicals that are found naturaly in vivo as products of metabolism and are used advantageously in certain biological functions, including an important role in the ability of imune system cels to protect against invading species. However, sustained 60 elevated levels of free radicals can lead to uncontrolled and unwanted reactions betwen free radicals and vital macromolecules in cels. For instance, free radicals can atack and significantly alter the structure of DNA, which can lead to the development of cancerous cels (Seifried et al., 2007). There are many other mechanisms by which oxidative stres can contribute to incidences of chronic ilnes. Antioxidants can aleviate oxidative stres in the body by reducing hyper-reactive oxidative species, thereby restoring the chemical balance in the body (Seifried et al., 2007). It has been shown that grapes contain antioxidant compounds that have specific health beneficial properties (Prior & Gu, 2005; Karthikeyan et al., 2007). Grapes contain phenolic antioxidants, including resveratrol, anthocyanins, and flavanols, among others (Kim et al., 2006; Choi & Le, 2008). Consumption of these compounds is believed to reduce the risk of certain human ilneses, including cardiovascular disease and cancer (Fan & Lou, 2004; Zern et al., 2005). The seds of the grape are rich in the phenolic compounds that are believed to contribute to these health benefits. The present study was conducted to investigate the chemical composition, free radical scavenging capacity, and anti-proliferative activities of the cold-presed sed flours and oils of five diferent grape (Vitis vinifera) varieties: Chardonnay, Concord, Norton, White, and Ruby Red. The data obtained from this study wil be used to identify varieties high in health-beneficial phytochemicals with potential for use as functional food ingredients. 61 Materials and Methods Materials and Chemicals Cold-presed flours and oils from the five varieties of grape seds were provided by Botanical Oil Innovations Inc. (Spooner,WI). These sed flours were composed of the solid residues remaining after cold-presed sed oil production and miling. Galic acid, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH ? ), Folin-Ciocalteu (FC) reagent, 6-hydroxy- 2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and !-tocopherol were purchased from Sigma-Aldrich (St. Louis, MO). 2,2?-azobis (2-aminopropane) dihydrochloride (AAPH) was obtained from Wako Chemicals USA (Richmond, VA), and 30% ACS grade H 2 O 2 was purchased from Fisher Scientific (Fair Lawn, NJ). Ultrapure water was manufactured by Cayman Chemical Co. (Ann Arbor, MI) and used in al experiments. Cel culture media components were obtained from Invitrogen (Carlsbad, CA), and an ATP-Lite 1step Luminescence Asay System was obtained from Perkin-Elmer (Waltham, MA). Al other chemicals and solvents were of the highest commercial grade and used without further purification. Lipid Extraction Grape sed flour was ground in a standard household coffe grinder to a consistency able to pas through a 40-mesh sieve. Hexane was used to de-fat the sample at a ratio of 1 g flour to 9 mL hexane. The samples were centrifuged for 10 minutes at 3000 rpm. Vacuum filtration was used to collect the samples and remaining hexane was evaporated in a nitrogen evaporator. 62 Antioxidant Extraction The grape sed flour samples remaining after hexane extraction were extracted with three diferent solvents: 70% ethanol, 50% acetone, and 70% acetone with 0.5% (v/v) acetic acid. Samples were extracted in a ratio of 1 g to 10 mL (Parry et al., 2006). The samples were vortexed three times for 30 seconds each. The samples were left on a 3-D rotator for 18 hours in the dark, and centrifuged prior to decanting of the supernatant for testing. Grape sed oil samples were extracted with 90% methanol in a ratio of 1 g to 9 mL. The samples were vortexed for 4 minutes and placed in the centrifuge for 5 minutes at 1,000 x g. The supernatant was collected, and the oil sample was extracted with 90% methanol two more times, and the supernatants were combined. Fatty Acid Composition Prior to gas chromatographic analysis, volatile derivatives of faty acids were prepared by saponification followed by methylation. This conversion of free faty acids to faty acid methyl esters (FAME) was performed using a previously outlined laboratory procedure (Yu et al., 2002). The FAME samples were subjected to GC analysis to identify the faty acid composition of the grape sed oil samples, using a Shimadzu GC-2010 with an FID, an AOC-20i injector, and an AOC-20S autosampler (Columbia, MD). Helium was used as the carrier gas at a flow rate of 12.2 mL/min through a fused silica capilary column SPTM-2380 (30 m ! 0.25 mm with a 0.25 "m film thicknes). Injection volume was 1 ?L with a split ratio of 10:1. Oven temperature was initialy 136 ?C, increased by 6 ?C/min until 184 ?C where it was held for 3 min, then increased again by 6 ?C/min to a 63 final temperature of 226 ?C. The relative faty acid profile of each variety was determined by comparing sample peaks in triplicate to that of known standards in order to identify the components. Carotenoid/Tocopherol Composition The grape sed oil for high performance liquid chromatography (HPLC) analysis of carotenoids and tocopherols were prepared by saponification acording to a method by Kurilich & Juvik (1999) with modifications. One mL of ethanol with 0.1% BHT was added to 3 mL of oil extract. The samples were placed in an 85 ?C water bath for 5 minutes, after which 2 mL of 2 M KOH was added. The samples were vortexed and returned to the water bath for 10 minutes for saponification. Immediately after saponification, the test tubes were placed in an ice bath, and 1.5 mL of 3 M NaCl and 1.5 mL of methyl tert-butyl ether (MTBE) were added to each sample. Samples were vortexed and centrifuged at 700 g?s for 10 minutes. The supernatant was decanted to a new tube. The MTBE wash was repeated twice more, and the supernatants were combined. Deionized water was then used to wash the combined supernatants. The MTBE was evaporated and the residue was redisolved with 1 mL of MTBE (Kurilich & Juvik, 1999). HPLC analysis was completed with the prepared samples in triplicate to determine carotenoid and tocopherol composition using a Shimadzu LC-20AD model with SPD-20A UV detector (Columbia, MD). The wavelength of the UV detector was set at 450 nm. Mobile phase A was methanol/MTBE/water (81:15:4, v/v/v) and mobile phase B was methanol/MTBE (9:91 v/v). The separation was achieved using a linear gradient of 100% of A to 50% A and 50% B in 45 min, followed by 100% B for 10 min to wash the column, 64 and 100% A for 5 min to re-equilibrate prior to the next injection. The flow rate was 1.0 mL/min with a column temperature of 25 ?C and an injection volume of 30 ?L. Carotenoids and tocopherols were quantified using comparisons of HPLC retention time and peak area of known concentrations of purchased standards. Total Phenolic Content Folin-Ciocalteu (FC) reagent was used to determine the total phenolic content (TPC) of the grape sed flour and oil extracts following a previously described laboratory procedure (Yu et al., 2002). A 50 !L sample of each of the grape sed flour and oil extracts was mixed with 250 !L FC reagent, 750 !L 20% sodium carbonate, and 3 mL of Ultrapure water. After reacting for 2 h at ambient temperature, absorbance was read at 765 nm on a Thermo Spectronic Genesys spectrophotometer (Waltham, MA). A separate standard curve was created for each extraction solvent using galic acid. Results were reported as mg galic acid equivalents per g of grape sed flour or oil, and were measured in triplicate for each sample. Sample extracts were diluted in extraction solvent as necesary to fal within the range of the standard curve. Oxidative Stability Index (OSI) OSI values were determined using a 743 Rancimat Metrohm (Herisau, Switzerland). The oxidation reaction was carried out at 86 ?C with an air flow rate of 7 L/h, and 60 mL of deionized water in each of the measuring vesels. Samples were tested in 6 mL aliquots of the grape sed oil. Commercial canola oil was used as the control. 65 Oxygen Radical Absorbance Capacity (ORAC) Asay ORAC values were determined for grape sed flour extracts following a previously described protocol (Moore et al., 2005). Trolox standards were prepared in appropriate solvents matching the grape sed flour extract being tested. Al other reagents were prepared in a 75 mM sodium phosphate buffer (pH 7.4). A 225 !L aliquot of freshly made 8.16 x 10 -8 M fluorescein solution was mixed with 30 !L sample, standard or blank in a black 96-wel plate and pre-heated at 37 ?C for 20 minutes. After pre-heating, 25 !L of freshly made 0.36 M AAPH was mixed into each wel, and the fluorescence of the reaction mixture was measured using a Victor 3 multilabel plate reader (Perkin-Elmer, Turku, Finland) once every two minutes for two hours at 37 ?C, with an excitation wavelength of 485 nm and an emision wavelength of 535 nm. Sample trolox equivalents (TE) were estimated based on the area-under-the-curve (AUC) method used by Ou and colleagues (2001). Results were reported as ?mol of TE per g of defated, cold-presed grape sed flour, and were measured in triplicate for each sample. Hydroxyl Radical Scavenging Capacity (HOSC) Estimation The HOSC asay was also conducted using fluorescein as the fluorescent probe and a Victor 3 multilabel plate reader (Perkin-Elmer, Turku, Finland) acording to a previously reported laboratory protocol (Moore et al., 2006). In brief, 30 !L of sample standard or blank was reacted with 170 !L of 9.28 x 10 -8 M fluorescein, 40 !L of 0.1990 M H 2 O 2 , and 60 !L of 3.43 M FeCl 3 in black 96-wel microplates. The fluorescence of the reaction mixture was recorded approximately once every 260 seconds for up to seven hours at ambient temperature, with an excitation wavelength of 485 nm and an emision wavelength of 535 nm. Standards were prepared in appropriate solvents matching that of 66 the flour samples being tested. Fresh 9.28 x 10 -8 M fluorescein solution was prepared for each asay from stock solution and 75 mM sodium phosphate buffer (pH 7.4). Trolox equivalents (TE) of each sample were calculated following the same AUC method used for the ORAC TE estimation (Moore et al., 2006). Results were reported as micromoles of TE per g of defated, cold-presed grape sed flour, and were measured in triplicate in each sample. DPPH ? Scavenging Activity Free radical scavenging activity was determined acording to a previously reported procedure by Yu and colleagues utilizing stable 2,2-diphenyl-1-picrylhydrazyl (DPH ? ) radicals (2003). The reaction consisted of an equal mixture of freshly prepared 100 !M DPPH ? solution and grape sed extract, primed to a total volume of 200 !L in a clear, flat- bottomed 96-wel plate. The absorbance was measured at 515 nm every 1.5 minutes for 40 minutes and compared against a standard curve consisting of six dilutions of 6-hydroxy- 2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) betwen 10 !M to 35 !M using solvent as the blank. Measurements were taken using a Victor 3 multilabel plate reader (Perkin-Elmer, Turku, Finland). Results are presented as ?mol TE/ g flour or oil. Anti-proliferative Activity against HT-29 Cels The anti-proliferative test was adopted from a previously described experiment (Wang et al., 2007). The HT-29 human colorectal adenocarcinoma cel line was cultured in a humidified incubator at 37 ?C and 5% CO 2 . The cel culture media contained McCoy?s 5A media, 10% fetal bovine serum, and 1% antibiotic/antimycotic solution. Cels were grown to 95% confluence and then plated at 2,500 cels/wel in a black 96-well 67 viewplate for experiment. After 24 h, the growth medium was replaced with 100 !L of either the control or treatment medium. An ATP-Lite 1step Luminescence Asay System for Perkin-Elmer was used to determine the amount of cel proliferation prior to treatment and at 4, 24, 48, 72, and 96 h after the initial treatment. Plates were permited to equilibrate to room temperature for a period of 30 mins before the reading was taken. Immediately before the reading was taken on a Victor 3 multilabel plate reader, 100 ?L of reconstituted ATP-Lite 1step Luminescence was added to each wel. The cel media was replaced with a fresh treatment or control solution every 24 h. Treatment Media Preparation. To prepare the treatment solutions for anti- proliferative tests, grape sed oil extracts in 90% methanol were evaporated in the nitrogen evaporator at 40 ?C until only water and extract remained. These samples were freeze-dried to remove the water. The flour extracts in 70% ethanol were also evaporated in the nitrogen evaporator at 40 ?C. Dried extracts were then disolved in DMSO. Treatment media were prepared by mixing DMSO extracts and cel growth media, which were then pased through 0.2 ?m retrograde celulose syringe filters. Cels were treated with three diferent doses of grape sed flour and oils: 0.99, 2.0, and 9.9 mg grape sed part equivalent/ mL. Al treatment doses contained the same amount of DMSO, including the vehicle control. Statistical Analysis Each asay was conducted in triplicate, with data reported in tables and figures as mean ? standard deviation. Diferences in means betwen samples were determined using one-way ANOVA and Tukey?s post hoc analysis. Statistical analysis was performed using 68 SPS software for Windows (version rel. 10.0.5, 1999, SPS Inc., Chicago, IL). Statistical significance was defined at P ! 0.05. Results and Discussion Fatty Acid Composition As shown in Table 3.1, cold-presed oils from diferent varieties of grape seds have similar faty acid compositions, difering only slightly in their components. A number of faty acids have health-promoting atributes. Linoleic acid (18:2) is found at high levels in the diferent grape varieties, and studies have shown that consumption of it may lead to a decreased risk of ischemic stroke (Iso et al., 2002). Linoleic acid was measured in comparable quantities across the 5 types of grape seds, ranging from 64.89 to 75.22 g/100 g oil in the grape sed oils, and Concord grape seds were shown to contain the highest amount (Table 3.1). The linoleic acid data obtained fals within the range of previously demonstrated values of 50.1 to 77.8 g/100 g oil (Yu et al., 2005). It was determined that each grape variety had similar oleic acid (18:1) compositions, ranging from 14.85 to 22.99 g/100 g oil, showing a slightly higher level in the Ruby Red variety (Table 3.1). These results show that while each of the oils obtained from the diferent grape varieties had slightly diferent faty acid profiles, each faty acid was present in similar overal proportions in al of the diferent samples. Based on faty acid contents, no one grape sed oil stands out above the others as a beter functional food ingredient. 69 Table 3. 1 Fatty Acid (FA) Composition of the Studied Cold-Presed Grape Sed Flours (g/100 g oil)* FA Chardonnay Concord Norton Ruby Red White 16:0 7.24 ? 0/01 6.84 ? 0.01 7.62 ? 0.01 6.87 ? 0.01 7.25 ? 0.01 18:0 3.87 ? 0.01 2.79 ? 0.01 4.19 ? 0.01 4.64 ? 0.01 3.92 ? 0.01 20:0 0.19 ? 0.01 NA 0.19 ? 0.01 0.17 ? 0.01 0.17 ? 0.01 16:1 0.09 ? 0.01 NA 0.08 ? 0.01 0.05 ? 0.01 0.08 ? 0.00 18:1 19.79 ? 0.02 14.85 ? 0.01 16.79 ? 0.00 22.99 ? 0.00 15.96 ? 0.02 18:2 68.59 ? 0.01 75.22 ? 0.04 70.71 ? 0.01 64.89 ? 0.01 72.35 ? 0.01 18:3 0.22 ? 0.01 0.29 ? 0.01 0.42 ? 0.01 0.19 ? 0.01 0.26 ? 0.01 *Values are based on triplicate readings. Data were expresed as mean ? SD (n=3). SFA: saturated faty acids. MUFA: monounsaturated faty acids. PUFA: polyunsaturated faty acids. na: not analyzed. Carotenoid/Tocopherol Composition The carotenoid and tocopherol contents of the grape sed oils are summarized in Figure 3.1. The grape sed oils were analyzed for !-tocopherol and two carotenoids: lutein and "-carotene. Both carotenoids and tocopherols are antioxidants. The oils from the various grape seds were evaluated for their !-tocopherol contents. Al oil samples contained !-tocopherol, with the lowest value detected in the Concord grape sed oil at 70 10.65 ?g/g oil, and the highest value detected in the Ruby Red grape sed oil at 87.09 ?g/g oil. Lutein and !-carotene were also detected in al of the grape sed oils. The consumption of lutein has also been linked to an increase in pigmentation in the macular region of the retina, which decreases the risk for certain eye diseases, including light- induced retinal damage and age-related macular degeneration (Landrum & Bone, 2001). Lutein was found to be the most abundant carotenoid compound present in the grape sed oils for al five varieties. Specificaly, the greatest lutein content was found in the extract of the White grape sed oil at 41.67 ?g/g (Figure 3.1). These results are comparable to a previous study that also reported lutein as one of the primary carotenoids found in grapes (Guedes de Pinho et al., 2001). In contrast, !-carotene was found to be the least abundant of the measured lipophilic antioxidants. The oil from al five grape sed varieties contained both carotenoid compounds, with the Norton grape sed oil sample having the highest total carotenoid content at 68.84 ?g/g, followed by the oil of the White grape sed at a total carotenoid content of 60.32 ?g/g. The White grape sed oils contained the greatest diference of carotenoids with 41.67 ?g /g oil of lutein and 18.65 ?g/g oil of !-carotene. The Concord grape sed oil contained the least amount of both lutein and !-carotene, at 7.39 and 4.23 ?g/g oil respectively (Figure 3.1). There was no correlation betwen carotenoid and tocopherol contents. Grape sed oils containing higher carotenoid amounts did not necesarily correlate to higher "- tocopherol amounts. For example, Ruby Red grape sed oil was found to have the highest amount of "-tocopherol, yet contained one of the lowest amounts of carotenoid compounds. Concord grape sed oil was found to have the lowest amounts of both 71 carotenoid and !-tocopherol compounds. The data indicates that grape seds of diferent varieties generaly contain varying levels of carotenoids and !-tocopherol, but al varieties do contain at least some of both carotenoids and tocopherols. Based on carotenoid and tocopherol data, diferent grape seds could be used as a functional food ingredient; the Ruby Red and Chardonnay varieties for !-tocopherol, and Norton and White varieties for carotenoids. Figure 3. 1 Composition of !-tocopherol and carotenoids in grape sed oils. Values are expresed as micrograms of analyte per gram of grape sed oil. Mean and standard deviation are shown (n = 3). The black boxes represent !-tocopherol, grey boxes represent lutein, and white boxes represent beta-carotene. For each analyte, values marked with the same leter do not difer significantly (n = 3; P " 0.05). b e d a c l m k l j y z v x w 0 10 20 30 40 50 60 70 80 90 100 Chardonnay Concord Norton Ruby Red White C h e mi c al C omp os i ti on (u g/ g of oi l ) alpha-Tocopherol Lutein (Xanthophyll) beta-Carotene 72 Total Phenolic Content The TPC values of each grape sed flour extract are shown in Figure 3.2. The TPC of the grape sed oil extracts were tested, but levels were too low to be quantified. Chardonnay grape sed flour showed the highest level of total phenolics in al extraction solvents, followed by Ruby Red, and White varieties. Concord and Norton contained the lowest total phenolics and contained statisticaly the same amounts for al three solvents. Grape sed flour samples extracted with 50% acetone exhibited the highest levels of phenolics; the greatest was Chardonnay with a TPC value of 129.8 mg galic acid equivalent (GAE)/g flour. Norton exhibited the lowest TPC value at 17.4 mg GAE/g flour when extracted with 50% acetone (Figure 3.2). Extraction with diferent solvents yielded difering estimations of phenolics. Chardonnay sed flour extracted with 50% acetone, as discussed above, had significantly greater level of phenolics (129.8 mg GAE/g flour) than Chardonnay sed flour extracted with 0.5% acetic acid in 70% acetone (105.9 mg GAE/g flour). The solvent, which consistently extracted the lowest levels of phenolics, was 70% ethanol, with a TPC value of 74.5 mg GAE/g flour for Chardonnay seds. Varying levels of phenolics with diferent extraction solvents indicate 0.5% acetic acid and 70% ethanol are not extracting phenolics present as wel as 50% acetone (Figure 3.2). White or light grape varieties have been shown previously to have a higher total phenolic content than red or purple grape varieties (Pastrana-Bonila et al., 2003). Chardonnay sed flour (a white grape) exhibited a TPC value of 186.3 mg GAE/g flour, which was higher than al other fruit seds in the study (Parry et al., 2006). Chardonnay 73 sed flours also contained the highest TPC of the 5 grape varieties in the present study. Grape sed oils are known to have low levels of phenolics, ranging betwen 0.06-0.1 mg GAE/g sed oil (Bail et al., 2008), which may explain why their levels were undetectable using the method of the current study. Yilmaz and Toledo (2006) showed the total phenolic content of grape seds was afected by extraction solvent. In their study, extraction with 50% acetone solution exhibited a greater TPC value than extraction with 70% methanol solution; a 0.5% acetic acid solution was not compared (Yilmaz & Toledo, 2006). Variations in phenolic content of grape sed flours can also result from variations in genetics and growing conditions. Based on TPC data, Chardonnay sed flour was a frontrunner candidate as a functional food ingredient. 74 Figure 3. 2 Total Phenolic Contents of Grape Sed Flours. Values were determined spectrophotometricaly using Folin-Ciocalteu reagent. Galic acid was used as the standard. Results shown as mg of galic acid equivalents (GAE) per g grapesed flour. The black columns represent an extraction solvent of 70% acetone with 0.5% (v/v) acetic acid, the grey columns represent 50% acetone, and the white represents 70% ethanol. For each solvent listed, values marked with the same leter do not difer significantly (n = 3; P ! 0.05) Oxidative Stability Index (OSI) Grape sed oil from the Norton, Ruby Red, White, and Concord varieties showed a significantly increased Oxidative Stability Index (OSI) compared to the canola oil control. OSI is a measurement taken of oils under acelerated oxidation conditions?it is the time at which an oil experiences the maximum change in the rate of oxidation. A higher OSI value (i.e., a longer time to reach the maximum change in the rate of oxidation) is a d d b c j m m k l v x x w w 0 20 40 60 80 100 120 140 160 Chardonnay Concord Norton Ruby Red White TP C (mg G A E/ g fl ou r ) Acetic Acid Acetone Ethanol 75 indicative of a beter shelf life. It is an indicator for the durability of the oil during storage (American Oil Chemist Society (AOSC), n.d.). This is an important factor when considering the shelf life of our product. As sen in Figure 3.3, the Norton and Ruby Red varieties exhibited the highest OSI. To make realistic comparisons, canola oil was used as a comparative control, as this oil is commonly used. OSI is determined by a variety of properties inherent to a food, including presence of polyunsaturated faty acids (PUFA). OSI is inversely related to oil?s PUFA content, as unsaturated fats are more easily oxidized. OSI is also determined by the tocopherol content of the oil, and in general more tocopherol is correlated with a higher OSI value, though the relationship is not linear (Tappel, 1998). The data indicated that Ruby Red oil has the lowest PUFA content and high tocopherol content, we observed a relatively high OSI value for Ruby Red grape sed oil. Norton, which had a low PUFA content and relatively low tocopherol content, had a high OSI value. The OSI value for the Chardonnay variety was unexpectedly low, faling below that of canola oil despite its low PUFA content (which was lower than that of the Norton variety) and high tocopherol content. 76 Figure 3. 3 Oxidative Stability Index (OSI) of grape sed oils. The OSI for the cold-presed grape sed oils of each variety and canola oil control were measured and the hours until oxidation are recorded. For each analyte Antioxidant Properties Al grape sed flour extracts exhibited scavenging capacities against peroxyl (ORAC) and hydroxyl (HOSC) radicals. Chardonnay grape sed flour was consistently present in the highest statistical group for antioxidant activity in every solvent tested (P ! 0.05) (Figures 3.4 & 3.5). For HOSC in 70% ethanol and 50% acetone, Chardonnay exhibited the highest radical scavenging values of 2,212 and 2,774 ?mol trolox equivalents (TE)/g defated sed flour, respectively (Figure 3.4). For ORAC 70% ethanol and 0.5% acetic acid in 70% acetone, Chardonnay exhibited the highest TE values of 1,429 and 1,131 ?mol TE/g defated sed flour, respectively (Figure 3.5). In the 50% acetone samples, Ruby Red exhibited a value of 2013 ?mol TE/g defated sed flour in c d c a a b 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Canola Chardonnay Concord Norton Ruby Red White O S I (h ou r s ) 77 comparison to 1,864 ?mol TE/g for Chardonnay; however, the two were not statisticaly diferent at the P ! 0.05 significance level. ORAC and HOSC values of Chardonnay found are higher than that of previously reported values for Chardonnay sed flour (Parry et al., 2006; Yilmaz & Toledo, 2006), but are similar to ORAC values reported for seds of Turkish grape varieties (Bozan et al., 2008). Figure 3. 4 Hydroxyl radical scavenging capacity of the cold-presed grape sed flour extracts. Values were measured against a trolox standard and expresed as ?mol of trolox equivalents (TE) per g of defated sed flour. Mean + SD values are shown (n = 3). The black column represents an extraction solvent of 50% acetone, and the grey represents an extraction solvent of 70% ethanol. For each solvent listed, values marked with the same leter do not difer significantly (n = 3; P ! 0.05) a c c b b j m m k l 0 500 1000 1500 2000 2500 3000 3500 Chardonnay Concord Norton Ruby Red White H O S C (mmol TE/ g fl ou r ) Acetone Ethanol 78 Figure 3. 5 Oxygen radical scavenging capacity of cold-presed grape sed flour extracts. Values were measured against a trolox standard and expresed as ?mol of trolox equivalents (TE) per g of defated sed flour. Mean + SD values are shown (n = 3). The black columns represent an extraction solvent of 70% acetone with 0.5% (v/v) acetic acid, the white columns represent an extraction solvent 50% acetone, and the grid represents an extraction solvent 70% ethanol. For each solvent listed, values marked with the same leter do not difer significantly (n = 3; P ! 0.05) Al grape sed flour extracts also exhibited DPPH ? Scavenging Activity. As sen in Figure 3.6A, Chardonnay extract exhibited the highest activity in al three solvents with 8.23 mmol (0.5% acetic acid), 9.21 mmol (50% acetone), and 6.49 mmol TE/g of flour (70% ethanol), followed by Ruby Red and White, then Concord and Norton. Activities exhibited by Ruby Red and White extracts were similar to each other. Concord and Norton had the lowest activity, and were similar to each other. The 50% acetone and 0.5% acetic a c d cd b j k l j k v y y w x 0 500 1000 1500 2000 2500 Chardonnay Concord Norton Ruby Red White O R A C ( ! mol TE/ g fl ou r ) Acetic Acid Acetone Ethanol 79 acid extracts were also comparable to each other, while the ethanol extracts exhibited significantly lower DPPH ? scavenging activity for al grape varieties. Of the grape sed oil extracts in methanol, Concord exhibited the highest activity with 11.08 !M TE scavenged per g of oil (Figure 3.6B). The remaining grape sed oil extracts were statisticaly similar, ranging from 3.09 to 5.92 !M TE/g of oil. The DPPH ? scavenging activity for Chardonnay grape sed flour has previously been identified to be 1.51 mmol TE/g of flour in 50% acetone (Parry et al., 2006). The approximate three to five fold diference can partly be atributed to the specificity of cultivars (De Beer et al., 2003), as wel as potentialy diferent growing conditions. Other reported DPPH ? scavenging activity includes the activity of Norton and Concord grape skins, at 0.9 mmol of TE/g and 0.8 mmol of TE/g, respectively (Munoz-Espada et al., 2004). Grape sed flour extract in acetic acid for Norton (2.51 mmol TE/g) and Concord (2.61 mmol TE/g) were greater than that of grape sed skins while the respective grape sed flour extracts from ethanol were fairly similar. Overal, the data have implication in improving extraction of antioxidants from natural sources and the potential health properties of diferent types of grapes and their by-products, specificaly the Chardonnay variety, as a food ingredient. 80 Figure 3. 6A DPPH ? scavenging activity of grape sed flour. Samples were evaluated for direct scavenging activity of DPH radicals and compared to a trolox standard. Results are shown as mean ? SD in units of milimoles of trolox equivalents (TE) per gram of defated grape sed flour. The black columns represent 70% acetone with 0.5% (v/v) acetic acid, the white columns represent 50% acetone, and the grey represents 70% ethanol. a b b a a j l l k k v y z w x 0 2 4 6 8 10 12 Chardonnay Concord Norton Ruby Red White R D S C (mmol TE/ gr am of fl ou r ) Acetic Acid Acetone Ethanol 81 Figure 3.6B. DPH Radical Scavenging capacity of grape sed oils. Samples were evaluated for direct scavenging activity of DPH radicals and compared to a trolox standard. Results are shown as mean ? SD in units of micromoles of TE/gram of grape sed oil. The black columns represent 90% methanol. Columns in a series marked by the same leter are not statisticaly diferent (n = 3; P ! 0.05). Anti-proliferative Activity against HT-29 Cels The results of the anti-proliferation HT-29 cel study show that the variety and concentration of grape sed flour and oil dictate the amount of cels that remain after treatment over time. The flours and oils, in three diferent concentrations, were compared with a vehicle control containing the same concentration of DMSO and were treated in the same manner. In each of the flour and oil samples, the high concentration (9.9 mg grape sed part equivalent/mL) of al of the grape treatments resulted in the greatest amount of cel inhibition after 96 h. For the Concord flour and oil, Chardonnay flour, Ruby flour, and White flour and oil, the number of cels actualy decreased (P < 0.05) over the entire 96 h in the high concentration of treatment. This shows that these types of extract and variety b a b b b 0 2 4 6 8 10 12 14 Chardonnay Concord Norton Ruby Red White R D S C ( ? mol TE / g of oi l ) 82 of grapes give a high value of anti-proliferative efects for these cancer cels. The vehicle control was used to show the exponential growth of the cancer cels given the media and sera combination used, and was used for comparison of the growth of treated cels. The high concentration treatments, 9.9 mg equivalence/mL, of White, Ruby, and Chardonnay flours virtualy eliminated the cancer cels within those samples after 48 hours, showing an extremely high level of efectivenes. These varieties also showed decreased growth of cancer cels in the low (0.99 mg grape sed part equivalent/mL) and medium (2.0 mg grape sed part equivalent/mL) treatment concentration levels when compared to the vehicle solution. The oils were les efective in slowing the growth of the cancer cels, but were stil able to decrease the amount of cel growth when compared to the vehicle control. As sen in Figure 3.7, the flours of White, Ruby, and Chardonnay grapes were the most efective and statisticaly significant in the anti-proliferation asay. This tentatively supports previous findings where treatments of 3 mg grape sed part equivalents/mL and 6 mg grape sed part equivalents/mL of Chardonnay grape sed flour extract was found to have nearly eliminated HT-29 cancel cels after 24 hours of treatment (Parry et al., 2006). The treatments in the present study were not as dramaticaly efective, but this stil confirms the clear trend that grape sed extract inhibits the growth of these cells. To evaluate the potential utilization of these grape sed flours and oils in cancer prevention, it is necesary to complete more evaluative tests on other cancer cel lines, as wel as normal cel lines. However, the results here suggest that Chardonnay, Ruby Red, and White are the most bio-active and are therefore have the most as potential functional food ingredients. 83 Figure 3.7 Time and Dose Effects of Grape Sed Flour and Oil Extracts on HT-29 Cel Proliferation. Relative luminescence is proportionate to the number of viable cels. Values are based on triplicate tests, with mean values show (n = 3). (A) Chardonnay Flour (B) Chardonnay Oil (C) Ruby Red Flour (D) Ruby Red Oil (E) White Flour (F) White Oil. The diamond represents the low treatment of 0.99 mg grape sed part equivalents/mL, the (A) (B) (C) (D) (F) (E) 84 square represents the medium treatment of 2.0 mg grape sed part equivalents/mL and the triangle represents the high treatment of 9.9 mg grape sed part equivalents/mL. Vehicle is represented by a circle. Conclusion About 66 milion tons of grapes were produced world wide in 2007; of these fresh grapes, 86.6% were further procesed into wine, jams, and grape juice (Maier et al., 2009). Grape seds are a waste product of the juice and wine industry, which generates large quantities of waste (5?9 milion tons per year, worldwide), which can sep into the ground and increase the chemical oxygen demand (COD) and the biochemical oxygen demand (BOD). Changing these characteristics of the natural environment can have detrimental efects on the flora and fauna of the waste discharge zones. Finding another use for grape seds could help to ameliorate this environmental problem (Bonila et al., 1999; Schieber et al., 2001; Louli et al., 2004). The results from this study indicate that grape sed flours and oils may serve as dietary sources of natural antioxidants that contain anti-proliferative properties, particularly Chardonnay grape sed flour. Additional research is necesary to further analyze the efects of food formulation, procesing, and storage on the availability of the beneficial properties. 85 Chapter 4: Chemical Composition and Health Properties of Whole Soft Wheat Bread Enriched with Cold-Presed Chardonay Grape Seed Flour and Oil The present study was conducted to investigate bread baked with the grape sed flour and oil at diferent times and temperatures, for their chemical composition, free radical scavenging capacities, and anti-proliferative activity against cancer cels. The data obtained from this study was used to determine the refined time and temperature that a functional food, such as bread, that has been incorporated with the grape sed components can stil exhibit the maximum amount of available health beneficial properties. Introduction The human body functions by maintaining metabolism through carefully monitored oxidation-reduction reactions. Oxidative stres can shift the balance of these reactions, causing elevated levels of free radicals in the body (Hennig et al., 2007). Chronicaly elevated levels of free radicals can lead to uncontrolled reactions with vital macromolecules (Seifried et al., 2007). Grapes and grape seds contain phenolic antioxidants that have been shown to contain health-beneficial properties by mitigating oxidative stres in the body caused by hyper-reactive oxidative species (Kim et al., 2006; Seifried et al., 2007; Choi & Le 2008). However, 67% of American adults consume les than two servings of fruit a day (Blanck et al., 2005). Grapes as fruits are not always available, afordable, or a practical choice for consumers. A functional food model alows 86 consumers to obtain additional antioxidants in a les perishable, economical product that is readily available. Bread has been previously chosen as the functional food model for other fruit additives due to its ease of preparation and status as a common food product (Fan et al., 2006). During procesing, specificaly baking, changes in chemical composition and food matrix alter the composition of antioxidants in a functional food model (Delgado-Andrade et al., 2010). Availability of these antioxidants may be afected by variation of food procesing conditions such as baking time and temperature, either positively or negatively as new compounds are formed or others are released from the food matrix (Moore et al., 2009). Original concentrations in raw ingredients and changes during incorporation into a functional food also afect bioavailability of factors critical for health-beneficial efects. The changes may be a result of chemical and biochemical reactions during storage and ingredient procesing and food preparation, as wel as interactions with other components during digestion (Kim et al., 2006). Bioavailability can vary drasticaly during digestion. In one case following both oral and gastric steps the amount of soluble antioxidants increased by approximately 17 fold while insoluble antioxidants decreased by approximately 10 fold (Delgado-Andrade et al., 2010). Results of eficacy trials involving bioavailable antioxidants also fluctuate wildly. Studies have shown in rats that antioxidants administered with a dry diet can predictably inhibit development of carcinogen induced cancer while antioxidant extracts administered with sesame oil did not (Kim et al., 2004). The present study was conducted to ases the availability (in vitro) of antioxidants, radical scavenging capacity, and eficacy of a grape sed bread functional 87 food model made with cold-presed Chardonnay grape sed flour and oil. Data from this study wil be used to determine optimal baking time and temperature as wel as viability of grape sed bread as a source of antioxidants. Materials and Methods Materials and Chemicals Cold-presed chardonnay grape sed flour and oil were provided by Botanical Oil Innovations Inc. (Spooner,WI). Galic acid, 2,2-diphenyl-1-picrylhydrazyl radical (DPH ? ), Folin-Ciocalteu (FC) reagent, 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid (Trolox), !-carotene, and "-tocopherol were purchased from Sigma- Aldrich (St. Louis, MO). 2,2?-azobis (2-aminopropane) dihydrochloride (AAPH) was obtained from Wako Chemicals USA (Richmond, VA), and 30% ACS grade H 2 O 2 was purchased from Fisher Scientific (Fair Lawn, NJ). Ultrapure water was manufactured by Cayman Chemical Co. (Ann Arbor, MI) and used in al experiments. Cel culture media components were obtained from Invitrogen (Carlsbad, CA), and an ATP-Lite 1step Luminescence Asay System was obtained from Perkin-Elmer (Waltham, MA). Al other chemicals and solvents were of the highest commercial grade and used without further purification. Baking Proces The bread samples were baked using a standard recipe and food-grade ingredients. First 118 g lukewarm water at approximately 27 ?C was added to 4 g sugar and 6 g yeast. This mixture was alowed to sit for ten minutes to proof the yeast. Meanwhile, the dry 88 ingredients were mixed separately; this included 4 g salt, 33 g sugar, and 88 g soft whole wheat flour. For the control product, 88 g of bread flour was also added to this mixture. For the grape sed product, 44 g bread flour and 44 g grape sed flour was added to the mixture. In addition to the unbaked control samples, control and grape sed bread samples were baked for each of the following conditions: 163 ?C for 30 minutes, 191 ?C for 20 minutes, 191 ?C for 30 minutes, 191 ?C for 40 minutes, and 218 ?C for 30 minutes. Each sample was prepared and baked in triplicate. Lipid Extraction Freeze-dried bread samples with and without grape sed flour were ground in a standard household coffe grinder to pas through a 40 mesh sieve. Hexane was used to extract the fat from the bread samples at 1g sample to 2 mL hexane ratio at ambient temperature for 5 minutes. The samples were centrifuged for 10 minutes at 3000 rpm. The hexane extraction was collected and stored at -16 ?C for further analysis. Antioxidant Extraction The defated bread samples were extracted with 50% acetone at 1 g of sample to 4 mL solvent ratio. They were vortexed three times for 30 seconds each and then left in a 3- D rotator for 18 hours in the dark. The supernatants were decanted and stored at -16 ?C until further analysis. 89 Lutein/Tocopherol Composition Sample Preparation. In order to prepare the lipid bread extracts for high performance liquid chromatography (HPLC) analysis, 1 mL of methyl tertiary butyl ether (MTBE) was added to a test tube containing 3 mL of the extract. The test tube was sealed and placed in a sonicator for 6 minutes. 0.2 mL of the sample was extracted from the test tube, and filtered through a 0.45 ?m filter. The preceding steps were completed for al of the 36 samples (Darnoko et al., 2000). HPLC Analysis. The HPLC system used to determine the lutein and tocopherol composition of the bread samples was a Shimadzu LC-20AD model SPD-20A UV detector EZstart system with a C-30 column with a length of 250 mm and a particle size of 5 !m. The wavelength for the detection was set at 450 nm for luteins and 295 nm for tocopherols. Mobile phase A was methanol/MTBE/water (81:15:4, v/v/v) and mobile phase B was methanol/MTBE (9:91, v/v). The gradient elution was as followed: 100% A to 77.8% A/ 22.2% B from 0 to 20 minutes, 100% B from 20 to 30 minutes, and re- equilibration of the column at initial gradient conditions from 30 to 40 minutes. The flow rate was set at 1.0 mL/min, and 30 ?L of each sample was injected into the HPLC system using the auto sampler. Identification and quantification of luteins and tocopherols were based on comparisons betwen HPLC retention time, and area under the curve of the sample peak with that of the standards (Darnoko et al., 2000). Total Phenolic Content Folin-Ciocalteu (FC) reagent was used to determine the total phenolic content (TPC) of the 50% acetone extractions of the grape sed flour enriched and control breads 90 following a previously described laboratory procedure (Yu et al., 2002). A 50 !L sample of each of the extracted breads was diluted and mixed with 250 !L FC reagent, 750 !L 20% (m/v) sodium carbonate, and 3 mL of Ultrapure water. After reacting for 2 hours at ambient temperature, absorbance was read at 765 nm on a Thermo Spectronic Genesys spectrophotometer (Waltham, MA). The standard curve was created using diferent concentrations of galic acid and results were reported as miligrams of galic acid equivalent per gram of bread. Oxygen Radical Absorbance Capacity ORAC values were determined following a previously described protocol (Moore et al., 2005). Trolox standards and bread extractions were diluted to appropriate amounts in 50% acetone. Al other reagents were prepared in 75 mM sodium phosphate buffer (pH 7.4). 225 !L of freshly made 8.16 ! 10 -8 M fluorescein solution was mixed with 30 !L sample, standard or blank in a 96-wel plate and pre-heated at 37 ?C for 20 minutes. After pre-heating, 25 !L of freshly made 0.36 M AAPH was added to each wel, and the fluorescence of the reaction mixture was measured using a Victor 3 multilabel plate reader (Perkin-Elmer, Turku, Finland) once every two minutes for two hours at 37 ?C, with " Ex = 485 nm and " Em = 535 nm. Sample trolox equivalents (TE) were estimated based on area- under-the-curve (AUC) method used by Ou et al., (2001). Results were reported as micromoles of TE per gram freeze-dried bread sample. 91 Hydroxyl Radical Scavenging Capacity The HOSC asay was conducted using fluorescein as the fluorescent probe and a Victor 3 multilabel plate reader (Perkin-Elmer) acording to a previously reported laboratory protocol (Moore, Yin, Yu, 2006). 30 !L sample or solvent was reacted with 170 !L of 9.28 ! 10 -8 M fluorescein, 40 !L of 0.1990 M H 2 O 2 and 60 !L of 3.43 M FeCl 3 in microplate wels, and the fluorescence of the reaction mixture was recorded approximately once every 260 seconds for three hours at ambient temperature, with " Ex = 485 nm and " Em = 535 nm. Trolox standards and bread samples were diluted to appropriate concentrations in 50% acetone. Fresh 9.28 ! 10 -8 M fluorescein solution was prepared for each asay from stock solution and 75 mM sodium phosphate buffer (pH 7.4). Trolox equivalents (TE) of each sample were calculated following the same AUC method used for the ORAC TE estimation (Moore et al., 2006). Results were reported as micromoles of TE per gram of freeze-dried bread samples. DPPH ? Scavenging Activity Free radical scavenging activity was determined acording to a previously reported procedure by Cheng et al., (2006) utilizing stable 2,2-diphenyl-1-picrylhydrazyl (DPH) radical. The reaction consisted of an equal mixture of freshly prepared 100!M DPH ? solvent solution and grape sed extract, primed to a total volume of 200 !L. The absorbance was measured at 515nm and compared against a standard curve consisting of six dilutions of 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid (Trolox) betwen 10 !M to 35 !M. The reaction was measured against the blank of solvent at room 92 temperature for 40 min using a Victor 3 multilabel plate reader (Perkin-Elmer). Breads baked in triplicate were also measured in triplicate. Anti-Proliferative Activity against HT-29 Cels Anti-proliferative activity against HT-29 cels was determined acording to a previously reported procedure by Wang and others (2007) and Slavin and others (2009). HT-29 human colorectal cels were cultured in cel media containing McCoy?s 5A media, 10% fetal bovine serum, and 1% antibiotic/antimycotic solution. The cels were placed in a humidified incubator at 3 ?C and 5% CO 2 , and grown to 95% confluence, and then plated at 2,500 cels/wel in a black 96-wel viewplate for tisue culture. After 24 hours, the medium was replaced with 100 !L of either the control or treatment medium. The amount of cel proliferation was determined prior to treatment, and at 4, 24, 48, 72, and 96 hours after treatment using an ATP-Lite 1-step Luminescence Asay System for Perkin- Elmer. Plates were permited to equilibrate to room temperature for a period of 30 minutes before the reading was taken. Immediately before the reading was taken on a Victor 3 multilabel plate reader, 100 ?L of reconstituted ATP-Lite 1step Luminescence was added to the wels. The cel media was replaced with a fresh batch of treatment or control solution every 24 hours until the plate was read. Treatment Media Preparation. To prepare the treatment solutions, the 50% acetone extracts of the grape sed enriched and control bread were first evaporated in the nitrogen evaporator to remove acetone, and then freeze dried overnight to remove the water. Samples were then disolved in DMSO. DMSO solutions were mixed with normal growth media to reach a concentration of 10 mg bread equivalents/mL for each sample, 93 and pased through a 0.2 ?m retrograde celulose syringe filters. Cels were treated with bread extracts cooked at varying times and temperatures, as wel as with an unbaked control. Statistical Analysis Each asay was conducted in triplicate, with data reported in tables and figures as mean ? standard deviation. Diferences in means betwen samples were determined using one-way ANOVA and Tukey?s post hoc analysis. Statistical analysis was performed using SPS software for Windows (version rel. 10.0.5, 1999, SPS Inc., Chicago, IL). Statistical significance was defined at P ! 0.05. Results and Discussion Lutein and !-Tocopherol Composition The bread samples were analyzed for their "-tocopherol and lutein contents. The tocopherol and lutein contents of the bread samples are summarized in Figure 4.1. The control bread samples contained higher "-tocopherol and lutein levels than the grape sed bread samples. This result may be due to the canola oil used in the control bread samples. Canola oil has been shown to have high levels of tocopherols, with "-tocopherol averaging 274.5 #g/g of oil in comparison to the 56.7 #g/g found in Chardonnay grape sed oil (Al- Saqer et al., 2003). Previous studies have also found canola oil to have high lutein levels (Farre et al., 2010). In addition, it is important to note that the standard deviation for the data was fairly high. This result is possible given the triplicate samples of bread were baked by hand, 94 which can lead to high variability in the data. Acording to the statistical analysis, the grape sed bread samples did not show significantly diferent tocopherol and lutein levels when compared to the control samples. Figure 4. 1 Lutein and !-Tocopherol Content of Control and Grape Sed Breads Based on Baking Conditions. Lutein and !-tocopherol in bread model baked at various times and temperatures compared to unbaked control bread. Solid columns represent !-tocopherol and open columns represent lutein. C indicates control bread with wheat and bread flour only. G indicates grape sed enhanced bread. The amount of !-tocopherol is consistently higher than the amount of lutein under varying baking conditions.Additionaly, the amount of !- tocopherol and lutein both vary depending on time and temperature. Data marked by diferent leters indicate significant diferences (P " 0.05), with a-c reflect the diference betwen tocopherol level, whereas j-l indicate the diference in lutein contents. a abc abc c abc bc bc abc ab bc abc bc j kl kl l kl l kl kl kl k kl l 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 ! - T oc op h e r ol an d Lu te i n ( ? g/ g of b r e ad ) alpha-Tocopherol Lutein 95 Total Phenolic Content The TPC values for grape sed and control bread at each baking condition are shown in Figure 4.2. Grape sed bread, baked and unbaked, had significantly higher levels of phenolics than control bread. Varying baking conditions had no statisticaly significant efect on phenolics in control bread. Grape sed bread baked at 163 ?C for 30 minutes had the highest level of phenolics with a TPC value of 25.8 mg galic acid equivalent (GAE)/g bread and showed a statisticaly significant diference from breads baked at 191 ?C for 20 and 40 minutes and at 218 ?C for 30 minutes (P < 0.05). Grape sed bread baked at 191 ?C for 40 minutes exhibited the lowest TPC value at 18.6 mg GAE/g bread and had a statisticaly significant diference from unbaked grape sed bread, bread baked at 163 ?C for 30 minutes and 191 ?C for 30 minutes (Figure 4.2). These results suggested that higher temperature may negatively afect the level of phenolics present in the grape sed bread, with longer baking time showing a slightly greater negative influence on TPC values, but this diference is not statisticaly significant from the highest temperature. The large standard deviations, especialy in grape sed bread baked at 163 ?C and 191 ?C for 30 minutes, were likely a result of variation in specific loaves of bread during the baking proces. Al loaves were not baked simultaneously and were not freeze-dried together. Grape sed enrichment has been found to increase the overal antioxidant activity of bread (Peng et al., 2010). Baking conditions have not been shown to significantly afect the total phenolic content of whole wheat piza crust (Moore et al., 2009). However, the total phenolic content of grape sed extracts exposed to heat treatment has 96 been found to increase with moderate cooking time and temperature, and decrease with heat high cooking time and temperature (Kim et al., 2006). These trends found in studies on grape sed extracts correlate with the trend founds when testing the grape sed bread in the current study. Figure 4. 2 Total Phenolic Contents of Grape Sed and Control Breads by Baking Conditions. The total phenolic content was determined spectrophotometricaly using Folin-Ciocalteu reagent. Galic acid was used as the standard. GAE mg/g is the mg of galic acid equivalents per g bread. Grape sed bread baked at 163 ?C for 30 minutes had the highest total phenolic content, while bread baked at 191 ?C for 40 minutes demonstrated relatively low phenolic content. Solid columns represent wheat bread controls and open columns represent grape sed bread. Data marked by diferent leters indicate significant diferences (P ! 0.05). j j j j j j ab a bc ab c bc 0 5 10 15 20 25 30 35 Unbaked 163 ?C, 30 min 191 ?C, 20 min 191 ?C, 30 min 191 ?C, 40 min 218 ?C, 30 min TP C (mg G A E/ g b r e ad ) Control Grape Seed 97 Antioxidant Properties Supplementing whole wheat bread with Chardonnay sed flour was found to significantly increase its scavenging capacities against peroxyl (ORAC) and hydroxyl (HOSC) radicals (P ! 0.05). Antioxidant capacity was increased by over 1000% in some of the bread samples with the substitution of grape sed flour for a portion of the wheat flour. In addition, baking time and temperature was shown to significantly afect ORAC and HOSC values for bread samples containing grape sed flour, but did not significantly change the antioxidant activities of non-supplemented bread samples(P ! 0.05) (Figures 4.3-4.4). The highest ORAC value of 628.6 ?mol TE/g dried sample was measured for the grape sed containing bread baked at 163 ?C for 30 min (Figure 4.3). In contrast, the highest HOSC value of 434.1 ?mol TE/g dried sample was observed for the grape sed supplemented bread baked at 191 ?C for 30 min (Figure 4.4). Overal, both radical scavenging capacity asays found antioxidant capacity of the bread increased by moderately lengthening the baking time, but extended baking times reduced the measured level of antioxidant capacity, although the diference in the ORAC asay was not statisticaly significant. The experimental results suggested that bread supplemented with grape sed flour and oil had high potential application for developing a functional food rich in natural antioxidants. In 2004, Wu et al. measured ORAC of common foods consumed in the United States. Grain and cereal products exhibited radical scavenging capacities of 10-20 ?mol TE/g fresh weight, consistent with that found in the control bread samples in this study (25-38 "mol TE/g freeze-dried weight). Foods that contained particularly high 98 levels of radical scavenging capacities, such as blueberries, artichokes, kidney beans, and pecans, al exhibited ORAC values betwen 100-200 ?mol TE/g fresh weight (Wu et al., 2004). The grape sed bread exhibited ORAC values betwen 410-630 !mol TE/g freeze- dried weight. Even though values reported in the current study were per freeze-dried sample weight, as opposed to fresh-weight basis, the determined ORAC values are stil congruent with added health beneficial properties of grape sed bread. HOSC values showed similar support of high antioxidant levels in grape sed supplemented bread. In comparison to the values measured in this study, 21-35 !mol TE/g of control bread, Moore and others found HOSC values for whole soft wheat and hard wheat bran to be 38.78 and 74.91 ?mol TE/g, respectively (Moore et al., 2006). 99 Figure 4. 3 Oxygen Radical Absorbance Capacity of Bread Extracts. Values were measured against a trolox standard and expresed as ?mol of trolox equivalents (TE) per g of defated sed flour. Mean + SD values are shown (n = 3). Solid columns represent wheat bread controls and the open columns represent grape sed bread. Values marked with the same leter do not difer significantly (n = 3; P ! 0.05). j j j j j j b a c c c b 0 100 200 300 400 500 600 700 800 Unbaked 163 ?C, 30 min 191 ?C, 20 min 191 ?C, 30 min 191 ?C, 40 min 218 ?C, 30 min O R A C ( ? mol TE/ g d r i e d b r e ad ) Control Grape Seed 100 Figure 4. 4 Hydroxyl radical scavenging capacity of bread extracts. Values were measured against a trolox standard and expresed as ?mol of trolox equivalents (TE) per g of defated sed flour. Mean + SD values are shown (n = 3). Solid columns represent wheat bread controls and open columns represent grape sed bread. For each solvent listed, values marked with the same leter do not difer significantly (n = 3; P ! 0.05) Al bread samples exhibited DPH radical scavenging activity (RSC). Al control breads exhibited significantly lower RSC ranging from 0.03 mmol TE/ g of bread to 0.101 mol TE/g of bread as shown in Figure 4.5. Bread containing grape sed flour baked at 163 ?C for 30 minutes exhibited the greatest RSC at 2.073 mmol TE/g bread, a value that was significantly higher than al other breads (P ! 0.05). Grape sed bread baked at 191 ?C for 20, 30 and 40 minutes and at 218 ?C for 30 minutes were not statisticaly diferent from each other comparable with respective RSC values of 1.517, 1.107, 1.395 and 1.544 mol TE/g of bread. Additionaly the grape sed flour breads baked at 191 ?C for 30 and j j j j j j b b b a b b 0 50 100 150 200 250 300 350 400 450 500 Unbaked 163 ?C, 30 min 191 ?C, 20 min 191 ?C, 30 min 191 ?C, 40 min 218 ?C, 30 min H O S C ( ? mol TE/ g d r i e d b r e ad ) Control Grape Seed 101 40 minutes showed similar RSC to the unbaked bread. The heat treatment during the proces of baking appeared to have released additional antioxidants, especialy at the lowest temperature combination of baking at 163 ?C for 30 minutes. It was possible that baking at higher temperatures and longer times subsequently destroyed the antioxidant components initialy released. RSC values of 2.4 !mol TE/g of bread and 2.1 !mol TE/g of bread on a per dry weight basis were previously reported for wheat bread (Michalska et al., 2007; Delgado-Andrade et al., 2010). The control wheat bread in the current study was ten times higher than these previously reported values. The use of diferent varieties, the specific use of soft wheat flour as opposed to hard wheat, as wel as the use of whole wheat in the current study are likely contributors to the observed diference in RSC. Some additives that have been utilized to enrich bread include bran and rye. The RSC of wheat bread enriched with bran (which more closely approximates the whole wheat flour used here) was stil lower than the current data at a reported 3.2 !mol TE/g of bread. This was stil almost 10 times lower than the control bread and 500 times les than the grape sed bread reported here (Delgado-Andrade et al., 2010). Rye bread also exhibited a similar RSC of 4.9 !mol TE/g bread (Michalska et al., 2007). Overal, bread made with grape sed flour and soft whole wheat contained significant levels of antioxidants, which implied it might have potential as a functional food for augmenting daily antioxidant intake. 102 Figure 4. 5 DPH Radical Scavenging Capacity of Bread Extracts. Solid columns represent control bread, and the open columns represent grape sed bread. Samples were evaluated for direct radical scavenging capacity (RSC) of DPH radicals and compared to a trolox standard. Results are shown as mean +/- SD in units of mmol of trolox equivalents (TE) per gram of defated bread sample. Columns in a series marked by the same leter are not statisticaly diferent (n = 3; P ! 0.05). Anti-Proliferative Activity against HT-29 Cels The results of the anti-proliferation HT-29 cel study showed that the treatments with grape sed bread extract had significant suppresed cel proliferation. While bread samples that were baked at diferent times and temperatures showed slightly, but not statisticaly significantly diferent results anti-proliferative activities. Anti-proliferative activity of the bread extracts did not sem to be dependent on time and temperature of baking. The proliferation of cels with treatments of varying baking temperatures is shown in Figure 4.6, which displays the efects of breads baked at various temperatures for 30 l k l l k j c a b bc bc b 0.00 0.50 1.00 1.50 2.00 2.50 Unbaked 163 ?C, 30 min 191 ?C, 20 min 191 ?C, 30 min 191 ?C, 40 min 218 ?C, 30 min R D S C (mmol TE/ g d r i e d b r e ad ) Controls Grape Seed 103 minutes. Al of the grape sed containing samples, including the unbaked bread dough sample, showed significant anti-proliferative activity as compared to the vehicle control. Slight diferences in cel proliferation were sen across time and temperature combinations of the grape sed bread extracts. However, the diferences betwen the grape sed bread treatments were not statisticaly significant. Al grape sed bread treatments showed statisticaly significant anti-proliferative activity when compared to the vehicle at 96 h. The proliferation of cels with treatments of varying baking times is shown in Figure 4.7, which shows the efects of breads baked at 191 ?C for varying lengths of time. The grape sed containing samples again significantly depresed the cel growth, as can be sen by comparing the cel growth of these samples to the cel growth of the vehicle. Again, diferences were sen in the relative number of cels surviving after treatment; however, the diferences betwen the grape sed bread treatments were not statisticaly significant. The grape sed treatments? inhibition of cel growth was statisticaly significant when compared to the vehicle treatment at 96 hours. The anti-proliferative activity of baked and unbaked grape sed bread treatments is compared to that of baked and unbaked control bread in Figure 4.8. The growth displayed by cels treated with control bread extracts baked at 191 ?C for 30 min was very high, exceding the growth curve of the vehicle treatment, which had only DMSO and media. The unbaked control bread behaved oddly, showing activity roughly similar to the vehicle or slightly les until the 96 hr reading; when the amount of cels dropped significantly so that the luminescence reading was lower than that of the other samples compared here. The baked grape sed sample, which was also baked at 191 ?C for 30 min, showed 104 antiproliferative activity and was significantly diferent when compared to the baked and unbaked control bread samples and the vehicle. The unbaked grape sed break extract semed to inhibit the cel growth slightly stronger than the baked grape sed sample, although the diference is not significant. These results indicated the grape sed contained active components inhibiting the cel growth, rather than other components in the bread. Figure 4. 6 Anti-Proliferative Effects of Grape Sed Bread Extracts by Baking Temperatures. Relative luminescence is proportionate to the number of viable cels. Values are based on triplicate tests, with mean values shown (n = 3). Al samples were baked for 30 minutes. ?X? with grey dash line represents the sample baked at 163 ?C, ?X? with dashed and one dot line represents the sample baked at 191 ?C, circle with dashed line represents the sample baked at 218 ?C, triangle with solid line represent the unbaked sample, and the square with dash and two dot line represents the vehicle 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 24 48 72 96 R e l ati ve Lu mi n e s c e n c e Hours Vehicle Unbaked 163 191 218 105 Figure 4. 7 Anti-Proliferative Effects of Grape Sed Bread Extracts by Baking Times. Relative luminescence is proportionate to the number of viable cels. Values are based on triplicate tests, with mean values shown (n = 3). Al samples were baked for 163 ?C minutes except for the unbaked control. ?X? with dashed line represents the sample baked at 20 minutes, ?X? with dash and one dot line represents the sample baked at 30 minutes, circle with dashed line represents the sample baked at 40 minutes, triangle with solid line represents the unbaked sample, and the square with dash and two dot line represents the vehicle. 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 24 48 72 96 R e l ati ve Lu mi n e s c e n c e Hours Vehicle Unbaked 20 min 30 min 40 min 106 Figure 4. 8 Proliferation of Cels With Grape Sed Bread and Control Bread Treatments at 48 Hours. Relative luminescence is proportionate to the number of viable cancer cels. Solid columns represent wheat bread controls and open columns represent grape sed bread. Values marked with the same leter do not difer significantly (n = 3; P ! 0.05). jk j k k k jk a a a a a a 0 50 100 150 200 250 300 350 Unbaked 163 ?C, 30 min 191 ?C, 20 min 191 ?C, 30 min 191 ?C, 40 min 218 ?C, 30 min R e l ati ve Lu mi me c s n c e Control Grape Seed 107 Conclusion In conclusion, this study demonstrated that grape seds may be incorporated into a functional food model. The grape sed containing bread showed stronger antioxidant activity and greater amounts of health beneficial compounds than did the bread containing solely wheat. Additionaly, the time and temperature of the baking proces may afect the levels of antioxidant activity and health beneficial components in the finished grape sed containing bread. However, the diferences sen betwen time and temperature combinations were smal compared to the diferences sen betwen the grape sed bread and the control (wheat only) bread. By incorporating health beneficial properties , such as those from grape seds, into bread, there may be potential to increase the amount of antioxidants consumed by the population. Further research is necesary to determine consumer aceptance and wilingnes to purchase such a product. 108 Chapter 5: Consumer Aceptability of Functional Bread Containing Cold-presed Grape Sed Oil and Flour Introduction Functional food products are defined as having health beneficial properties beyond basic nutrition (International Food Information Council, 2002). Interest in functional foods has grown in the past decade as a result of higher health care costs, and growing evidence indicates that diet is directly related to the development of chronic human diseases (Milner, 2000). Since functional food products have the potential to improve health while reducing the incidence of disease, they have become an atractive mainstay in the consumer market. Functional food and beverage sales in the US were $24.8 bilion in 2006, with sales estimated to increase by 56% to $38.8 bilion in 2011 (Zawistowski, 2010). In this study, grape sed oils and flours were incorporated into bread to create a functional food product. Grape seds have been shown to have many health-beneficial properties, most notably their antioxidant properties. Antioxidants can minimize the efects of oxidative stres by eliminating reactive oxidative species in the body (Limaset et al., 1993). This elimination is desirable because increased oxidative stres has been linked to a higher incidence of chronic ilneses, including heart disease and cancer. Despite the health benefits of antioxidants, research indicates that many Americans do not consume enough fruits and vegetables?valuable sources of antioxidant 109 components?in their diet. In 2009, the Centers for Disease Control and Prevention reported that only 32.5% of adults consumed the recommended fruit intake of two or more fruits daily, and only 26.3% of adults consumed the recommended vegetable intake of three or more vegetables daily (Grim et al., 2010). Since fruits and vegetables can be rich sources of antioxidants, these numbers suggest that many Americans may not be receiving an adequate amount of antioxidants in their diet. Bread was chosen as a functional food model because it is a widely-consumed and highly acesible food product that could potentialy increase antioxidant consumption for a large American population. However, it was stil uncertain whether grape sed bread would be acepted in a consumer market, since health beneficial properties alone may not be enough to determine the consumer aceptability of a functional food. Previous research indicated that consumer aceptability of new functional food products is based on both their awarenes of health benefits and favorable sensory qualities (Verbeke, 2006). Grape sed bread is a novel functional food model, as such litle previous work has been done to understand the consumer response to it. Therefore, the purpose of this study was to measure potential consumer aceptance of a functional food product containing grape sed flour and oil. Specificaly, sensory evaluation was conducted by freshman university students to determine whether there would be significant preferences towards the taste, texture, and color of control bread versus the grape sed bread. 110 Methods Sample Preparation Al samples were prepared and baked one to two days before the sensory analysis was conducted. First, the ?wet? ingredients were mixed; 118 g lukewarm water at approximately 27 ?C was added to 4 g sugar and 6 g yeast. This mixture was alowed to sit for ten minutes to proof the yeast. Meanwhile, the dry ingredients were mixed separately: 4 g salt, 33 g sugar, and 88 g wheat flour. For the control bread, 88 g bread flour was also added to this mixture. For the grape sed product, 44 g bread flour and 44 g grape sed flour was used. This ?dry? mixture was then placed in a standing mixer and mixed on medium. After the dry ingredients had been thoroughly mixed, the wet ingredient mixture was added, continuing to mix on medium speed. Additionaly, 8 g oil was added to the dough; for the control sample, this was canola oil, and for the grape sed sample, grape sed oil was added. These ingredients were mixed for approximately 3-5 minutes, or until the dough had an even consistency throughout. The dough was then removed from the mixer and placed on a cutting board lightly coated with whole wheat flour. The dough was kneaded by hand for 1-2 minutes, and then placed in a lightly oiled bowl; oil used for the bowl was congruent with the type used in the bread. The bowl was then covered with a dampened paper towel and placed into a warm oven. After alowing the dough to rise for one hour, the bowl was taken out of the oven and the dough was punched down. Then, the bowl with the dough was returned to the warm oven to rise for another 30 minutes. 111 Once the rising stage was complete, the dough was removed from the warming oven and transferred into bread pans for baking. The bread dough was then placed in a preheated oven at a temperature of 191 ?C. The bread was baked for a total of 30 minutes; after the bread had been in the oven for 25 minutes, the top of the bread was sprayed lightly with water, and then returned to the oven for the remaining 5 minutes of baking. The baked bread was removed from the oven after 30 minutes of baking and placed on a cooling rack for 10-20 minutes. The bread was then covered in plastic wrap and stored at room temperature for sensory analysis the following day. Sensory Analysis Sensory analysis was conducted in GEMS100 Freshman Honors Colloqium: Introduction to Gemstone clases at the University of Maryland, College Park. Subjects were, therefore, freshman in the Gemstone Program (n = 78). Al trials were conducted in the same room. Tasting stals were created using blank poster board displays to block the view of adjacent participants. For each stal, a smal cup of water and soda crackers were provided, as wel as a paper plate with two diferent bread samples, labeled A and B. The grape sed bread sample was labeled A, and the control bread sample was labeled B. The physical setup for the sensory analysis is shown in Figure 5.1. 112 Figure 5. 1 Diagram of the sensory analysis setup. Each clas of approximately 8-10 students came separately to the testing room. After students sat down at their tasting booth, a script of instructions was read to them (se Apendix A). The instructions described the nature of the experiment and directed them to carefully read the consent form, which was approved by the University of Maryland Institutional Review Board (se Apendix B) to decide if they wanted to participate in the study. Once participants read and completed the form, they were instructed to taste both breads, cleaning their palate with the soda crackers and water betwen each sample. The participants were also instructed to fil out a survey (se Apendix C), stating their preference betwen the samples, and rating each sample on color, texture, and flavor. The participants were not told which bread sample contained grape sed ingredients. The favorability rating was measured on a horizontal line of length 10.3 cm, on which participants could mark their preference with a single vertical line; a mark at the beginning 113 of the line, at 0 cm, indicated a strong dislike for the sample, and a mark at the end of the line, at 10.3 cm, indicated a strong like for the sample. There was also a smal section for comments about their sample preference. At the conclusion of the study, the surveys were collected, and a brief explanation of the study was provided. Data Analysis The preferences indicated on each survey were consolidated; the markings of the participants were measured so a numerical value could be asigned to each response. This number was the length measurement from the left end of the line on the survey in centimeters. Again, a smal number corresponds to disfavor while a larger number corresponds to favor. Lastly, the values from al the surveys for each individual question were averaged and the standard deviation was determined. Statistical analysis was performed on the mean preference ratings for color, texture, and taste for the grape sed and control breads. The test run was a paired 2-tailed Student's T-test, with pairing betwen the responses made by each student. Diferences in means betwen samples were determined using one-way ANOVA. Statistical analysis was performed using SPS software for Windows (version rel. 10.0.5, 1999, SPS Inc., Chicago, IL). Statistical significance was defined at P ! 0.001. 114 Results In this study, there were 78 participants, al freshman college students at the University of Maryland, College Park. Of the 78 participants, 11 had no preference betwen the two samples, 53 preferred the control bread, and 14 preferred the grape sed bread. The results for the mean preference ratings for taste, texture, and color, as wel as their standard deviations, can be found in Table 5.1. These results indicate that participants preferred the color, texture, and flavor of the control over the grape sed bread. The responses had large relative variations, with %CV of 35.5-44.7 % for grape sed bread, and 23.5-26.5 % for control bread, for means betwen values of 5-10. However, because of the large sample size, the diference in means betwen grape sed bread and control bread were found be significantly diferent in al three categories of color, texture, and flavor (P < 0.001). While the majority of participants ranked the sensory properties of the control bread higher than that of the grape sed bread, overal both samples were aceptable, as measured by the mean preference values for each sample category. A value of 5.15 out of the 10.3 scale would indicate neutral favorability, while any values lower than 5.15 show disfavor. The distribution of expected mean preference values were estimated by calculation 95% confidence intervals for each sample, and in each case, was found to be higher than 5.15 (Se Table 5.2.). This indicates that in al subsequent sensory evaluations, the mean of the preference value for both grape sed and control bread in 115 each category of color, texture, and flavor would be expected to fal above neutral preference. To analyze the distribution of the data further, a box and whisker plot were created and presented in Figure 5.2. When comparing the lower quartile limit of the control sample to the median value of the grape sed bread for each sensory category of color, texture, and flavor, it can be sen that the control sample lower quartile limit increases relative to the grape sed bread median from texture, to color, to flavor. This suggests that among those surveyed, the preference for control over grape sed bread was the strongest in the case of texture, and weakest in the case of flavor. Additionaly, there are relatively few outliers in the box and whisker plot, showing that the mean values were not significantly skewed by the presence of outliers. 116 Table 5. 1 Mean Preference Ratings of Color, Texture, and Flavor for Grape Sed and Control Breads with %CV. Color Texture Flavor Grape Sed Bread 5.7 ? 2.0 (35.5%) 5.5 ? 2.4 (44.7%) 6.4 ? 2.5 (38.5%) Control Bread 7.1 ? 1.7 (23.5%) 7.7 ? 2.0 (26.5%) 7.8 ? 2.0 (25.2%) Each rating is out of 10.3, with higher ratings showing more favorability. Grape sed and control breads were statisticaly diferent (n=78, P=0.001). Table 5. 2 Color, Texture, and Flavor Mean Preference Interval at 95% Confidence Level for Grape Sed and Control Breads. Color Texture Flavor Grape Sed Bread 5.2-6.1 4.9-6.0 5.8-6.9 Control Bread 6.7-7.5 7.2-8.1 7.3-8.2 Each rating is out of 10.3, with higher rating showing more favorability. 117 Figure 5. 2 Box and Whisker Plot of Grape Sed Bread Sensory Analysis Data. The box indicates the interquartile range. Outliers are marked with asterisks. Outliers are calculated by determining if they were below Q 1 ? 1.5!IQR or above Q 3 + 1.5!IQ. There were 78 participants. Preferences were measured from a 0 to 10.3 cm scale. Conclusion The results indicated significant diferences betwen consumer aceptability of the control bread and the grape sed bread. More respondents preferred the sensory qualities of the control bread than the grape sed bread, with the most prominent diference noted in the textures of the two breads. Of the three categories evaluated, both the control and the grape sed bread received their highest rating in flavor. The diference in preference betwen the two breads was much smaler for flavor than the diference betwen the two 0 1 2 3 4 5 6 7 8 9 10 11 Flavor: Control Flavor: GS Bread Texture: Control Texture: GS Bread Color: Control Color: GS Bread Preference Units Q u e s ti on an d S amp l e T yp e !" !" !" !" !" !" Strongly Dislike Strongly Like !" 118 breads for texture. Despite the preference for control bread over grape sed bread, many of the respondents did indicate that they had an overal favorable perception of the grape sed bread. Mean preference values show that the grape sed bread scored above a neutral rating for al three sensory categories tested. Data from this study suggest that consumers might be wiling to consume a bread product containing grape seds; however, further research is required to determine if these results would be true for a larger population. 119 Chapter 6: Conclusion Three comprehensive studies were conducted to determine the feasibility of creating a functional food product rich in natural antioxidants that could be used to lower the risk of chronic ilneses in the general population. Grape seds were selected as the source of nutraceuticals, both for their high antioxidant activity and their current role as an agricultural by-product. Bread was chosen as the functional food model because it is commonly consumed by most socio-economic classes in the United States. In addition, the baking conditions for bread are easily modified to test the effect of procesing on the chemical composition and health beneficial properties of the functional food. Finaly, a sensory survey was used to evaluate the aceptability of the proposed functional food, and to determine if the addition of grape seed flour and oil significantly affected the appeal of bread to consumers. The first study quantified the health beneficial properties of seds from five diferent grape varieties: Chardonnay, Concord, Norton, Ruby Red, and White. Extractions were made from cold-presed grape seed flour and oil samples using four diferent solvents. Grape seed flours and oils were found to contain varying levels of antioxidant properties, chemical composition, and anti-proliferative activity against colon cancer cells. Of the grape varieties tested, Chardonnay exhibited the highest level of health beneficial properties, overal, and was chosen for incorporation into the bread model in the second study. In addition, 50% acetone was found to be the most efective solvent for 120 extracting phytochemicals from grape seds, and thus was used for subsequent chemical asays in the second study. The second study compared the health beneficial properties of incorporating grape seed flour and oil into a standard bread recipe with those of a control bread product. The addition of grape seed flour and oil increased the antioxidant activity of the bread by roughly 10-fold, as well as significantly improving its carotenoid and !-tocopherol contents. Antioxidant activity and chemical composition of the bread also varied with the baking time and temperature, although only grape seed bread treatments exhibited significant diferences betwen time and temperature combinations. However, altering cooking time and temperature did not significantly change the antioxidant profile in the control bread. The decrease in antioxidants in the grape seed bread, but not in the control could potentialy be atributed to less thermaly stable antioxidants present in grape seds in comparison to those found in wheat flour, although the possibility was not evaluated in detail. The sensory evaluation conducted in the third study analyzed consumer preference for control and grape seed breads in terms of appearance, taste, and texture. Preference for control bread over grape seed bread was found to be statistically significant for all 3 sensory factors surveyed. However, both the control bread and the grape seed bread had overal positive reviews, suggesting that consumers would be wiling to consume this novel functional food product. These results suggest that grape seed bread can potentialy become a viable commercial functional food product; however, further research is necesary to determine if the positive impresions detected here would translate into consumer purchases and consumption, particularly when weighing the product?s novel 121 sensory properties and possible increased cost against the available health-beneficial properties. There is great potential value in creating a grape seed bread commercial product. The incorporation of grape seed flour and oil into the bread recipe has been shown to significantly increase its potential health beneficial properties. In addition, because grape seds are currently a waste-product of juice and wine production, the cost to produce this functional food would be expected to be significantly lower than purchasing current ?superfoods? promoted as significant sources for natural antioxidants. This product would also increase the commercial value of grape seds, and thus benefit the agricultural community by increasing the overal value of grape production. However, before such a product could be sold to consumers, its safety and long term health effects should be evaluated. Limitations and Future Research Directions The main limitation and direction for further study was the lack of in vivo data directly supporting the health-beneficial properties that were measured for bread containing grape seed flour and oil. While extensive literature exists supporting the links betwen antioxidant compounds, chemical measurements for antioxidant activity, reduced oxidative stres in lab animals, promoting health in lab animals, and finaly promoting health in humans, the overal chain of thought is tenuous at best, and often claimed to only apply in a case-by-case proces due to the complexity of metabolism in the body. With such a study, the consumption of the grape sed enriched bread could be directly linked with reduction of biomarkers of chronic ilneses in the test subjects. However, the cost to run such an animal study or clinical trial was prohibitively expensive at this time. 122 A much simpler problem to tackle would be the small number of baking conditions evaluated in the second part of this study. While 5 covariate points were sufficient for individualy examining the effects of baking time and temperature, more would be beneficial to study the interaction betwen time and temperature. Additional points would have also increased the robustnes of the conclusions made regarding the effects of baking conditions on health beneficial properties of the bread. More replications of the existing 5 baking conditions would also improve the strength of the conclusions drawn from the results. Another direction for further study existed with the consumer sensory survey. The study makes conclusions based upon the asumption that first year college students will be representative of consumers with respect to their taste and perception when evaluating a novel functional food. However, the asumption was made out of necesity, because of the infeasibility of surveying a large sample of people across the spectrum of the population, including diferent ages, ethnic groups, and socioeconomic classes. However, all of these factors could conceivably alter a consumer?s perception of bread containing grape seds. These factors could be acounted for if the sample size were expanded, given more materials for baking bread, and time for finding suitable subjects and conducting sensory surveys. This study could have also evaluated other grape varieties. The grapes studied were limited by the grape seds provided externaly. However, with additional funding, more varieties could have been evaluated by purchasing grape seds from industry and renting machinery to cold-pres the seds to obtain flour and oil samples. This would 123 increase the agricultural and economical value of this research, especialy if the purchased grape varieties were the most commonly used by the juice and wine industry. Finaly, the feasibility of commercialy producing the novel bread product developed in this project could be further studied using a thorough cost-analysis. To predict the price of this product, the cost of securing the grape seds for the modification to a whole wheat bread recipe would have to be considered in conjunction with the chalenge of modifying existing infrastructure to cold-pres grape seds on an industrial scale and adding the resulting flour and oil to current bread production lines. 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Your participation is entirely voluntary, and there is no penalty for declining to participate. If you are interested in participating in our study, please thoroughly read and complete this consent form. [Hand out consent forms to students that wish to participate.] Please note that the samples you wil be asked to taste as part of this study contain wheat flour, grape sed flour, grape sed oil, vegetable oil, sugar, yeast, salt, and water. Please do not participate in this study if you have an allergy to bread or any of the ingredients or have a medical condition that prohibits you from consuming gluten. [Team IMMUNE member wil wait for students to read and sign consent form if they stil wish to participate, and then collect the papers and put them in a sealed envelope.] You wil be given two samples of bread. Betwen tasting the two different samples you should clean your palate by eating the cracker and taking a sip of water. For each sample, you wil circle an answer or rate your reactions by marking with a vertical tick mark betwen strongly dislike and strongly like scale line. Please mark with a clear line because they wil be measured for data collection. Please do not include your name or any identifying information on the survey. Please do not converse or interact with any other participants during tasting. If you have any questions regarding this study, feel fre to ask me at any time. You can choose to stop participating in this study at any time. [Team IMMUNE member wil then give each student two samples of bread. Sample A wil be the control bread sample; Sample B wil be the grape sed sample. Also, each participant wil be given a smal glas of water and table crackers. Once the participant has finished the survey, the Team IMMUNE member wil collect the surveys and place them in a separate sealed envelope.] Thank you for your time! Our Gemstone Team is in our last year of research. We have already completed a comprehensive chemical analysis of many different types 145 of grape sed flours and oils, and designed our own novel grape sed bread through baking many prototypes. We are now using chemical tests to asses the antioxidant properties of our bread, and are using these surveys to asses if the bread is acceptable to consumers. If you have any further questions regarding this study or our team?s project, please feel fre to contact me directly or e-mail our group at immune@umd.edu. [Any remaining bread samples, table crackers, or water glases wil be thrown away. The IMMUNE member wil then leave the clasroom and return the sealed envelopes to Dr. Yu?s office] 146 Page 1 of 2 Initials ____ Date ___ Appendix B: CONSENT FORM Project Title Consumer Aceptance Analysis of Grape Sed Bread Why is this research being done? This is a research project being conducted by the Gemstone IMUNE undergraduate research team in cooperation with the Laboratory of Nutraceuticals and Functional Fods at the University of Maryland, Colege Park. The purpose of this research project is to promote the adoption and utilization of grape seds integrated into daily diet. Thus, the objective of this experiment is to examine the consumer perception of prepared bread that contains grape sed flour and oil that are rich in antioxidants. What wil I be asked to do? The procedure involves your evaluation of a grape sed bread sample and a control wheat bread sample. You wil be given bread to taste and then asked to complete a survey about the bread?s taste, texture, and color. The entire procedure should take no longer than aproximately twenty (20) minutes. What about confidentiality? We wil do our best to kep your personal information confidential. To help protect your confidentiality, we wil not colect any identifying information on the surveys. Surveys wil be kept in a locked ofice. If we write a report or article about this research project, your identity wil be protected to the maximum extent posible. Your information may be shared with representatives of the University of Maryland, Colege Park or governmental authorities if you or someone else is in danger or if we are required to do so by law. What are the risks of this research? There may be some risks from participating in this research study. If you are alergic to any of the ingredients in the bread (wheat flour, bread flour, salt, sugar, water, yeast, oil, grape seed flour, grape seed oil), you should not participate in this study. If you have a medical condition that makes you unable to eat gluten products, you should not participate in this study. There are no other known risks associated with participating in this research project. 147 Page 2 of 2 Initials ____ Date ___ Project Title Consumer Aceptance Analysis of Grape Sed Bread What are the benefits of this research? The benefits to you include increased awarenes of the health-beneficial properties of antioxidants, specificaly in grape sed derivatives. Do I have to be in this research? May I stop participating at any time? Your participation in this research is completely voluntary. You may chose not to take part at all. If you decide to participate in this research, you may stop participating at any time. If you decide not to participate in this study or if you stop participating at any time, you wil not be penalized or lose any benefits to which you otherwise qualify. What if I have questions? This research is being conducted by Dr. Liangli Yu of the Department of Nutrition & Food Science at the University of Maryland, College Park. If you have any questions about the research study itself, please contactDr. Liangli Yu at: 303 Marie Mount Hall, Colege Park, MD 20740, (301)-405-0761, or lyu5@umd.edu. If you have questions about your rights as a research subject or wish to report a research-related injury, please contact: Institutional Review Board Ofice, University of Maryland, Colege Park, Maryland, 20742; (e-mail) irb@deans.umd.edu; (telephone) 301-405-0678 This research has ben reviewed acording to the University of Maryland, Colege Park IRB procedures for research involving human subjects. Statement of Age of Subject and Consent Your signature indicates that: you are at least 18 years of age; the research has ben explained to you; your questions have ben fully answered; and you frely and voluntarily chose to participate in this research project. NAME OF SUBJECT SIGNATURE OF SUBJECT Signature and Date DATE ! 148 Appendix C: Team IMUNE Survey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losary Anthocyanin: a common flavanoid which gives grapes and grape seds their purple coloring. Anthocyanin?s health benefits include anti-inflamation of blood vesels and reduction of platelet coagulability (Maza, 2007). Anticarcinogenic: having to do with preventing the development of cancer Antimycotic: antifungal Antioxidants: molecules that reduce oxidative stres in the body by slowing and preventing free-radical oxidation reactions, which occur naturaly in the human body anti- proliferative activity Atherosclerosis: the hardening of the arteries caused by formation of plaques in the arteries. It can lead to strokes and heart atacks. [Beta]-carotene: a carotenoid that is a precursor to vitamin A. It?s strongly red-orange colored and common in fruits and plants (i.e. carrots). Bioactive: having an efect on living tisue Bioavailability: physiological availability of a chemical in the body for use Biomarkers: biological molecules used as an indicator to measure progres of proceses Carotenoids: photosynthetic plant pigments that are fat-soluble and posses antioxidative properties. Examples include beta-carotene and lutein. Cold presed: temperature controlled procesing technique in which oil is produced with the use of low heat technique. High heat degrades the flavor and the nutritional value of the oil product, so cold presing yields higher quality. Cytotoxicity: toxicity to cels 150 Dietary fibers: parts of grains, fruit, and vegetables that contain celulose. They are not digested by the body and help the intestine to absorb water. Flavanoids: most common group of polyphenolic compounds in the human diet. They?re commonly found in plants and are also known as vitamin P. Key types of flavanoids present in grapes and grape seds are proanthocyanidins, anthocyanins, and resveratrol. Fre radicals: strong oxidizing molecules that exist in the human body as a natural product of metabolism and function Functional food: food that provides an additional physiological benefit beyond that of its inherent nutritional value (Bidlack et al., 2005) Glutathione (GSH): tripeptide that have an peptide linkage betwen the amine group of cysteine and the carboxyl group of the glutamate side-chain. It has antioxidative properties and can exist in both reduced (GSH) and oxidized (GSG) states. Glycation: bonding of sugar to a lipid of protein Homeostasis: the stable and normal state Lutein: a type of carotenoid that may reduce the risk of certain eye diseases (Landrum et al., 2001). Low-density lipoprotein (LDL): one type of carrier of cholesterol and fat in blood, linked to increased risk of heart disease Malignant: tending to get worse or spread Monocyte: a type of white blood cel and part of the human imune system. Nutraceutical: a food that has specific health benefits, combines the words "nutrition" and "pharmaceutical" Oxidative stres: imbalance betwen the reactive oxygen species and a biological system 151 to detoxify the reactive intermediates. It can be caused by overexposure to sunlight, smoking, daily exposure to certain chemicals, and improper diet. Plaque: buildup of white blood cel deposits on the inside wal of an artery Polyphenols: conjugated chemicals that have been shown to have antioxidative properties. They?re commonly found in plants and are also known as phenoloids, phenols, or phenolics. Relative luminescence: the amount of light emited, corresponds with the number of living cels Resveratrol: a type of polyphenol that is naturaly produced by plants. Saponification: chemical proces by which faty acids from tryglycerides are separated from glycerol to enable quantification by gas chromatography Tocotrienol: a member of the vitamin E family. Vitamin E is made up of four tocopherols and four tocotrienols. Vitamin E is an important antioxidant in the body. Unsaturated fatty acids: carboxylic acids with a long unbranched and unsaturated tail. They are unsaturated because they have one or more double bonds. Thrombosis: formation of a blood clot within a blood vesel