Fruit Juices: Ellagic Acid Concentration and Sensory Appeal

Home , Cranberry juice, Ellagic acid, Ellagitannin, Grape juice, Pomegranate juice

FRUIT JUICES: ELLAGIC ACID CONCENTRATION AND SENSORY APPEAL

Caroline Kamau

A Thesis

Submitted to Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTERS OF FAMILY CONSUMER SCIENCES

August 2007

Committee:

Julian H. Williford, Jr., Advisor

Dawn Hentges

Priscilla Coleman

ABSTRACT

Julian H.Williford, Jr, Advisor

Dietary polyphenols such as ellagic acid (EA) have been associated with decreased

incidence of oxidative-stress related disease. The objective of this study was to determine the

concentration of ellagic acid in selected fruit juices and their blends. A sensory evaluation was

conducted to determine the preferences, and overall acceptance of the juice blends based on

color, mouthfeel and sweetness. The juices were hydrolyzed using hydrochloric acid and then

analyzed for ellagic acid concentration using HPLC-UV. Pomegranate juice had the highest

concentration of ellagic acid, 103 mg/L, while the other juices ranged from 1 mg/L to 2 mg/L.

The 3:1 pomegranate-cranberry mixture had the highest EA concentration of 97 mg/L of all juice combinations.

Purple grape juice was a component in all of the blends that ranked high in all preferences and also rated highest in the overall acceptance. Pomegranate juice was in all the blends that ranked low in all preferences and also rated lowest in the overall acceptance. The results suggest that the juices that ranked or rated high in color, sweetness and mouthfeel did not have high concentrations of ellagic acid.

iii

This project is dedicated to my parents, Anderson and Margaret Kamau, my siblings Morrine, Edwin and Martin.

ACKNOWLEDGMENTS

It has been such an honor working with knowledgeable people throughout the

development of this thesis.

First, I thank my advisor, Dr. Williford for his advice, suggestions and other forms of support.

Thank you Dr. Hentges, for being so approachable, and for the wonderful and bright suggestions and ideas. Thank you to Dr. Coleman for the support and encouragement. Thank you to the

Statistical Consulting Center, especially Dr. Nancy Boudreau for the much needed help in statistical analysis. To all my panelists, thank you for your participation and also for the six

weeks commitment to the study.

A special thank you to Dr. Endres, for your relentless support, brilliant ideas and suggestions,

keeping me focused through out the project and above all being a good mentor and a friend.

Last but not least, I want to thank my parents Anderson and Margaret, my sister Shiru, brothers

Edwin and Martin for their love and constant encouragement. Thank you to my close friends for

the emotional and other forms of support.

And most importantly, thank you to God for blessing me with good health and renewing my

strength throughout this project.

To all of you, again thank you for making this project a success. I surely could not have gotten

through without you.

TABLE OF CONTENTS

Page

CHAPTER I. INTRODUCTION …………………………………………………………… 1

Significance of the Problem ………………………………………………………... 4

Statement of the Problem .…………………………………………………………... 5

Hypotheses of the Study …………………………………………………………… 6

Objectives of the Study ……………………………………………………………. 6

Assumptions of the Study …………………………………………………………. 7

Limitations of the Study ………………………………………………………….. ... 7

Definition of Terms ……………………………………………………………. …... 7

CHAPTER II. REVIEW OF THE LITERATURE ……………………………………….. 9

Chemical Structure of Ellagic Acid…………………………………………………. 10

Effects of Ellagic Acid in the Body…………………………………………………. 11

Antioxidants and Cancer…..….. ...………………………………………………….. 13

Health Benefits of Fruit Juices…….………………………………………………… 13

Apple Juice………….…….…………………………………………………. 13

Purple Grape Juice……….....……………………………………………….. 15

Pomegranate Juice…………..………………………………………………. 17

Cranberry Juice…………...…………………………………………………. 20

HPLC Analysis ………………..….………………………………………………… 21

Sensory Evaluation...…………………………...... 22

Taste Panels...…………………………….………………………………….. 23

Test Methods for Taste Panels...…...…..……….…………………………… 24

Selection of Panelists ……….…………….……………………………….. 24

Screening Procedures…….....…………….……………………………….. 25

Identification Test..……..…..…………….……………………… 25

Basic Taste Test.....……..…..…………….……………………… 25

Ranking Test……..……..…..…………….……………………… 25

CHAPTER III. MATERIALS AND METHODS……………………………………….... 26

Materials……… ...... 26

High Pressure Liquid Chromatography-UV analysis of ellagic acid...... 26

Sample Processing and Analysis…………………………………………… 27

Soluble Solids Content...... 27

Sensory Evaluation of the Juices...... 27

Taste Panelists...... 29

Participants’ Screening Procedures...... 30

Tasting Location ...... 32

Tasting Procedure ...... 32

Statistical Testing of Collected Data...... 33

CHAPTER IV. RESULTS AND DISCUSSION...... 34

Selection of Taste Panelists ...... 34

Sensory Testing of Juice Mixtures...... 35

HPLC-UV analysis ...... 48

Typical HPLC-UV Results…………………………………………………. 48

vii

CHAPTER V. SUMMARY AND CONCLUSION....………………………………….... 54

REFERENCES………… ...... 59

APPENDIX A. CONFIDENTIAL QUESTIONAIRE ...... 65

APPENDIX B. CONSENT LETTER...... 67

APPENDIX C. SENSORY SCREENING TEST...... 70

APPENDIX D. SENSORY EVALUATION SCORE CARD...... 74

APPENDIX E. HPLC ANALYSIS ...... 77

viii

LIST OF TABLES

Table Page

1 ºBrix values for the individual and combined juices ...... 28

2 Concentrations of the matching taste test solutions...... 30

3 Concentrations of the discrimination taste test solutions...... 30

4 Friedman test of the panelist’ ranking of color, sweetness, and mouthfeel...... 38

5 Concentration of ellagic acid in the individual juices and juice blends...... 52

6 Phenolic compound composition of pomegranate juices...... 57

7 Potential panelists screening test results...... 73

8 Hedonic rating scale test ...... 75

9 Replicate results of HPLC analyses of individual juices and juice blends...... 78

LIST OF FIGURES

Figure Page

1 Chemical structure of bound ellagitannin and free ellagic acid……………………. 11

2 Flowchart of juice combinations for sensory evaluation ...... 29

3 Flowchart of juice combinations for sensory evaluation...... 32

4 The mean of a hedonic scale test for overall acceptance of sweetness preference

of 18 different combinations of juices…………………………………………… ... 35

5 The mean of a hedonic scale test for overall acceptance of color preference of 18

different combinations of juices…………………………………………………… 36

6 The mean of a hedonic scale test for overall acceptance of mouthfeel preference

of 18 different combination of juices………………………………………………… 37

7 The mean of a ranking test for color preference of six different 1:1

combination of juices……………………………………………………………….. 39

8 The mean of a ranking test for color preference of six different 1:3

combination of juices……………………………………………………………….. 40

9 The mean of a ranking test for color preference of six different 3:1

combination of juices……………………………………………………………….. 41

10 The mean of a ranking test for sweetness preference of six different 1:1

combination of juices……………………………………………………………….. 42

11 The mean of a ranking test for sweetness preference of six different 1:3

combination of juices….…………………………………………………………… 43

12 The mean of a ranking test for sweetness preference of six different 3:1

combination of juices….…………………………………………………………… 44

13 The mean of a ranking test for mouthfeel preference of six different 1:1

combination of juices……………………………………………………………….. 45

14 The mean of a ranking test for mouthfeel preference of six different 1:3

combination of juices……………………………………………………………….. 46

15 The mean of a ranking test for mouthfeel preference of six different 3:1

combination of juices……………………………………………………………….. 48

16 HPLC chromatogram of ellagic acid from the ellagic acid standard and juice……. 49

17 Ellagic acid standards calibration curve…………………………………….……… 50

1 CHAPTER I

INTRODUCTION

Polyphenols are a broad family of natural compounds widely found in plant foods.

Phenolic compounds embrace a considerable range of substances that possess an aromatic ring bearing one or more hydroxyl constituents. Polyphenols can be divided into two subgroups; flavonoid and phenolic acids. Ellagic acid is a phenolic acid compound. Polyphenols are a class of phytochemicals found in high concentrations in many botanical types of nectar, especially in gentian, strawberries, raspberries, cranberry, grapes, pomegranate and their beverages like red wine, apple, purple grape juice, pomegranate and cranberry juices.

Phytochemicals or phytonutrients are compounds found in plants that are not required for normal functioning of the human body. Phytochemicals may have a beneficial effect on health or an active role in the amelioration of disease. Phytochemicals have many functions in the human body, such as promoting the function of the immune system, acting directly against bacteria and viruses, and reducing inflammation (Wada and Ou 2002). Phytochemicals are also associated with the prevention and reduction of risk from diseases, such as cancer, cardiovascular disease.

There is abundant evidence from epidemiological studies that the phytochemicals in fruits and vegetables can significantly reduce the risk of cancer, cardiovascular disease, and retard the progression of atherosclerosis (Miguel and others 2004). As a result, consumers want to know the amount of phytochemicals, such as ellagic acid, in the commercially available juices.

In addition to the vitamins and minerals known to be present in fruits and vegetables, phytochemicals, such as flavonoids and other phenolics, may contribute protective effects since they have antioxidant activity (Wada and Ou 2002). Consequently, phytochemicals work to repair and prevent the action of activated oxygen molecules, free radicals which include nitric

2 oxide, hydroxyl radical (HORAC), singlet oxygen, hydrogen peroxide, super-oxide radical, and the combination of super-oxide and nitric oxide called peroxynitrate, generated in the metabolic pathways, all of which may cause dysfunctions when they interact with various tissue components. These highly reactive compounds can act as initiators and/or promoters, cause DNA damage, activate procarcinogens, and alter the cellular antioxidant defense system. Free radicals can alter cholesterol in an oxidation process in the arteries speeding up the onset of atherosclerosis, which may lead to coronary artery disease. Antioxidants function as inhibitors at both the initiation and promotion stages of carcinogenesis and protect cells against oxidative damage (Sun 1990).

Epidemiological studies have demonstrated that the composition of phenol-rich foods retards the progression of arteriosclerosis and reduces the incidence of cardiovascular diseases by preventing oxidative stress, that is, lipid peroxidation in arterial macrophages and in lipoproteins

(Miguel and others 2004). Dietary phenolic compounds are also known to elicit vital cellular responses, such as cell cycle arrest, apoptosis and differentiation by activating a cascade of molecular events (Narayanan and others 2002).

In a study conducted with 10 healthy participants, there was substantial inhibition of platelet activity after drinking two cups of purple grape juice daily for one week. Drinking the same amount of orange or grapefruit juice resulted in no platelet inhibition. This study suggested that the phenolic compounds in the purple grape juice may be strong platelet inhibitors (Keevil and others 2000).

The phenolic phytochemicals are present in glycosidic and non-glycosidic forms. The glycosides are mainly confined to hydrophilic regions in the cells, such as in vacuoles and apoplasts probably because of their higher water solubility. Glycosylation of the hydroxyl groups

3 on the phenolic ring of a phenolic phytochemical renders the molecule more water-soluble and less reactive toward free radicals (Urquiaga and Leighton 2000). Glucose is the most commonly involved sugar in glycoside formation, although galactose, rhamnose, xylose and arabinose and disaccharides, such as rutinose, have also been reported to be present in plants (Urquiaga and

Leighton 2000). Polymeric phenolics, such as tannins, exist as condensed tannins and are formed by condensation of single catechins and flavonols. They are either soluble or bound to the cell wall. Hydrolysable tannins are esters of a sugar with either gallic acid (gallotannins) or ellagic acid (ellagitannins). Tannins have higher antioxidant properties than individual phenolics, are not bioavailable, and are anti-nutritive in their function because of their ability to bind and precipitate biological macromolecules, such as proteins and carbohydrates (Vattem and others

2005). The total phenolic phytochemical content in plant foods varies greatly. The phytochemical content in plant foods is largely influenced by genetic factors and environmental conditions. Nevertheless, factors, such as cultivar, variety, maturity, processing and storage, also may influence the content of phenolic phytochemicals (Vattem and others 2005).

Synergy is the ability of two or more functional components, such as antioxidants in a phytochemical, to mutually enhance their functionalities (Vattem and others 2005). Combination of resveratrol and quercetin exerts a synergic effect in the inhibition of growth and proliferation of human oral squamous carcinoma cells. Synergistic interactions between wine polyphenols, quercetin and resveratrol were found to decrease the inducible nitric oxide synthase (iNOS) activity in cell culture systems. In food plants, each phenolic phytochemical has its own mode of action against a particular target. These modes of actions could be due to their ability to function as classical antioxidants or because of their ability to modulate cellular physiology by disrupting membrane functions or by altering the redox balance and energy metabolism of the cell.

4 Nevertheless, when quercetin and resveratrol are present together their ability to function together rapidly improves the overall result of maintaining the cellular homeostasis (Vattem and others 2005). For example, a study was conducted to determine the synergistic interactions of ellagic acid and rosmarinic acid on enhancing antimutagenic properties in S. typhimurium tester system against mutagens sodium azide. In addition, the ability of ellagic acid and rosmarinic acid to protect oxidative damage to DNA was also investigated using super-coiled DNA strand scission assay. Results showed that ellagic acid was most effective in inhibiting the mutations in

S. typhimurium system; whereas, rosmarinic acid and ellagic acid were equally effective in protecting the DNA from oxidative damage. Consumption of blends of fruit juices with biologically active biphenyls or other fruit can impart unique functional attributes and could be a more effective strategy in developing diet-based management of oxidation linked diseases such as mutagen and DNA damage induced carcinogenesis (Vattem and others 2004).

Phytochemicals in fruits, vegetables, whole grains, and other plant foods can have complementary and overlapping mechanisms of action including antioxidant activity and scavenging of free radicals; regulation of gene expression in cell proliferation, cell differentiation, oncogenes, and tumor suppressor genes; induction of cell cycle arrest and apoptosis; modulation of enzyme activities in detoxification, oxidation, and reduction; stimulation of the immune system; regulation of hormone metabolism; and antibacterial and antiviral effects (Liu and others 2005).

Significance of the Problem

Phenolic phytochemicals, such as ellagic acid, contribute to human health and are important components of fruits and vegetables which are sometimes consumed in juices

(Rangkadilok and others 2005). Many epidemiological studies have researched the health

5 benefits of phytochemicals found in fruits and vegetables; nevertheless, more research needs to be conducted to determine how these vital compounds should be made more accessible to people by ensuring that they are in different types and varieties of foods. Educational programs that enable people to understand the importance of these compounds to their health should be developed and introduced to the public.

Statement of the Problem

A study was conducted in the year 2000 by the National Health Interview Survey (NHIS) aimed at providing intake estimates for fruits and vegetables among Americans based on various demographic and behavioral characteristics. According to this study, well-educated individuals, those engaged in other healthful behaviors, underweight, and normal weight individuals were found to have intakes closer to recommendations by the Food Guide Pyramid (Thomson and others 2005). With most people becoming aware of the importance of phytochemicals to their health, they want to know how they can incorporate more phytochemicals into their diet.

Increasing the consumption of fresh fruits and vegetables, and their 100% juices may be an option.

While mixing of juices is not a new practice in the food industry, a review of literature revealed no studies where mixing of juices was for the purpose of enhancing the ellagic acid content in the juice mixture. In light of the continuing need for effective anticancer and other chronic disease prevention agents, and the association of phytochemical compounds to provide protective effects on human health, as reported in the research literature (Fergurson and others

2004), the current study was conducted to determine the concentration of ellagic acid, in commercially available single strength fruit juices as purchased, and in combination with other juices that may contain higher, lower, or no concentration of these compounds in ratios of 1:1,

6 1:3, 3:1, (v/v). The purpose of this study was to determine better combinations of the selected juices that contained the highest concentration of ellagic acid, and also had consumer acceptable taste, color, flavor and mouthfeel.

Hypotheses of the Study

The hypotheses that were tested in this investigation were:

Ho1: A combination of pomegranate and cranberry juice will have a higher concentration of ellagic acid than each juice alone.

Ho2: Pomegranate juice will have the highest concentration of ellagic acid when compared to the other individual juices in this study.

Ho3: A combination of pomegranate and cranberry juice will be the most acceptable juice combination as identified by the sensory panel.

Ho4: Pomegranate juice will have the highest °Brix as compared to other juices in this study.

Ho5: The blend of juices that will be most acceptable to the panelists will also have the highest concentration of ellagic acid.

Objectives of the Study

Therefore, the objectives of this study were:

1. To determine the amount of ellagic in the selected single fruit juices.

2. To determine the ellagic concentrations of the fruit juice mixtures.

3. To enhance the ellagic content in the combined fruit juices.

4. To determine the consumer acceptability of the combined fruit juices by using a trained

sensory evaluation panel.

5. To develop a fruit juice beverage which has elevated ellagic acid content with

acceptable color, taste, flavor, and mouthfeel.

7 Assumptions of the Study

1. Taste panelists did not eat, drink (except water), or smoke one hour prior to the testing,

or wear perfumes or scented hygiene products during the taste testing period.

Limitations of the Study

1. Taste and color thresholds were tested only in specific juices and their blends, and

therefore, can only be generalized as such.

2. Panelists had no prior experience in sensory testing, and therefore, they had to be

trained before participating in the test.

3. Information obtained from the subject questionnaire was self-reported.

4. Most students were not willing to commit themselves to participating in the study for the six

weeks period thus making it harder to get enough taste panelists.

5. The retention time of the ellagic acid peaks was inconsistentant from session to session thus

limiting the accuracy of the results.

6. The ellagic acid standards precipitated few hours (5 > hours) after preparation, thus limiting

their use and fresh standards had to prepared each time.

Definition of Terms

1. Phytochemical are compounds found in plants that are not required for normal

functioning of the human body.

2. Synergy is the ability of two or more functional components such as antioxidants to

mutually enhance their functionalities.

3. ºBrix is a measurement of the mass ratio of dissolved sucrose to water in a liquid.

4. Polyphenols are a group of chemical substances found in plants, characterized by the

presence of one or more phenol group per molecule.

8 5. Chronic diseases are those that continue for a long time or are recurrent.

6. Hydrolyze breaking a bond in a molecule by adding water.

7. Isocratic HPLC elution are operations at a single, constant mobile phase composition.

8. Recognition threshold is the lowest concentration that can be recognized as one of the basic

tastes i.e. sweet, sour, bitter and salty.

9. Doubling time is how long it takes for prostate-specific antigen (PSA) levels to double, a

signal that the cancer is progressing.

9 CHAPTER II

REVIEW OF THE LITERATURE

The increased interest in improved health and phytochemicals’ benefits to the human body have led to more interest in understanding of the chemical compounds found in fruits and vegetables. Polyphenols are present in a variety of plants, such as tea, blackberries, raspberries, cranberries, grapes, apples, and pomegranates, which are utilized as important components in human diets.

Chronic diseases constitute a major challenge for medicine and will probably remain so for future decades (Urquiaga and Leighton 2000). Nevertheless, an emergence, in epidemic proportions, of modern chronic diseases occurred in the latter part of the 20th century, and the diseases, are a problem to human health if they remain in progress. Some of these diseases are associated with changes in diet and lifestyle which contribute to the development of chronic diseases. Among the risk behaviors contributing to these conditions are excessive dietary fat intake, sedentary life style, smoking, environmental pollution, lack of physical exercise, and low

intake of fruits ad vegetables (Urquiaga and Leighton 2000).

With the help of cutting edge technology, the primary focus of the health professionals is

the early detection and treatment of individuals at disease risk. Nevertheless, prevention is a

more effective strategy than is treatment of chronic diseases. Strikingly, there are some common

risk factors and pathophysiological conditions that are eminent in most diseases in the category

of chronic diseases, such as, cardiovascular disease, some forms of cancer, stroke, Alzheimer’s

disease, and some of the functional declines associated with aging (Urquiaga and Leighton

2000). Oxidative stress, the result of an imbalance of prooxidants and antioxidants in an

organism, is rapidly gaining recognition as a key phenomenon in developing chronic diseases.

10 Oxidative stress is directly involved in the pathogenic mechanism of risk factors and in the protection exerted by various environmental factors. Diet plays a major role in the environmental control of oxidative stress; fruits, vegetables red wine and other foods have phytochemicals that help decrease oxidative stress. Researchers have estimated that one third of all cancer deaths in the United States could be avoided through appropriate dietary modification (Liu 2003). In its

1982 report on diet and cancer, the National Academy of Sciences included guidelines emphasizing the importance of fruit and vegetables in the diet (NRC 1982). In 1989, another report on diet and health from the National Academy of Sciences recommended consuming five or more servings of fruit and vegetables daily to reduce the risk of cancer and heart disease

(NRC 1989). Consequently, the 5-A –DAY program was developed as a tool to increase public awareness of the health benefits of fruits and vegetable consumption (Liu 2003).

Chemical Structure of Ellagic Acid

Ellagic acid (EA) exists either in a free unbound form or bound as ellagitannins, (Figure

1). Hydrolysable tannins are molecules with a polyol (mostly D-glucose) as a central core. The hydroxyl groups of these carbohydrates are partially or totally esterified with phenolic groups, such as gallic acid or ellagic acid. These hydrolysable tannins are present in a rich variety of plants, as well as teas, red wines, fruits and juices (Soong and Barlow 2006). Hydrolysable tannins are easily hydrolyzed by hot water, enzymes, mild acids or bases releasing EA units.

Ellagic acid has been found to exhibit antimutagenic, antiviral, anticancer, antitumor, and antioxidant properties, along with whitening of the skin. A study conducted using mango kernel extracts, showed an increase in antioxidant activity with increasing severity of hydrolysis.

Methanolic extracts showed a 24% and 32% rise in acid-equivalent antioxidant capacity

(AEAC), respectively, after hydrolysis at 85°C. This suggests that some conjugated phenolics

11 might be released by acid hydrolysis, and the free form might provide more potent antioxidant activity (Soong and Barlow 2006).

Ellagitannin Ellagic acid

Figure 1. Chemical structure of bound ellagitannin and free ellagic acid (Source: Scalbert and Williamson 2000; Lee and others 2005)

Effects of Ellagic Acid in the Body

Ellagic acid is an effective antimutagen and anticarcinogen phytotherapeutic agent that prevents carcinogens binding to DNA and strengthens connective tissue, and thus may keep cancer cells from spreading, inhibiting cancer onset and tumor proliferation and protecting healthy cells during radiation therapy and chemotherapy (Falsaperla and others 2005). This is made possible by stimulating various gluthatione-S-transferase isoforms involved in cytodetoxifying processes, free radical scavenger action and inhibition of correlated lipoperoxidative damage (Falsaperla and others 2005). Ellagic acid acts as a scavenger and binds or chemically engages cancer-causing chemicals or cytotoxic substances making them inactive.

12 Ellagic acid may also form adducts with DNA, masking the binding site to be occupied by the mutagen or carcinogen (Falsaperla and others 2005).

Ellagic acid may not only protect healthy cells and reduce cancer and cytotoxic induced chromosome damage, but it may also enhance the apoptotic mechanism normally inhibited in cancer cells. In a study conducted by the Hollings Cancer Institute, ellagic acid was found to inhibit the mitotic phase of cancer cells and block the cells in G1/S transition phase, prevent gene p53 destruction by cancer cells, determine IGF-II down-regulation, activate gene p21

(waf1/Cip1) and enhance natural killer cell mediated antitumoral immune response (Narayanan and others 1999). In a related study conducted to investigate ellagic acid-induced cell cycle arrest and apoptosis in T24 human bladder cancer cells in vitro, ellagic acid significantly reduced the viable cells, induced G0/G1-phase arrest of the cell cycle and apoptosis. In addition, ellagic acid caused an increase of p53 and p21 and a decrease in CDK2 gene expression, which may lead to the G0/G1 arrest of T24 cells. Upon exposure of caspase-3 activity to ellagic acid for 1, 3, 6, 12 and 24 h, induction of apoptosis was observed (Li and others 2005).

A study was conducted to determine the potential cytotoxic and anti-proliferative activities of ellagic acid using human umbilical vein endothelial cells (HUVEC), normal human lung fibroblast cells HEL 299, Caco-2 colon, MCF-7 breast, Hs 578T breast, and DU 145 human prostatic cancer cells. Ellagic acid (1-100µmol/L) inhibited HUVEC tube formation and proliferation on a reconstituted extra cellular matrix and showed strong anti-proliferative activity against the colon, breast, and prostatic cancer cells (Losso and others 2004). Ellagic acid induced cancer cell death by apoptosis and also induced reduced cancer cell viability demonstrated by decreased ATP levels of the cancer cells (Losso and others 2004).

13 Antioxidants and Cancer

Evidence suggests that dietary antioxidants can reduce cancer risk. In an epidemiologic review of approximately 200 studies conducted by Block and colleagues (1992) the relationship between fruit and vegetable intake and cancers of the lung, colon, breast, cervix, esophagus, oral cavity, stomach, pancreas, and ovary were examined. In 128 of 156 dietary studies, the consumption of fruit and vegetables was found to have a significant protective effect. The risk of cancer for most cancer sites was twice as high in persons whose intake of fruit and vegetable was low compared to those with high intake. Significant protection with high fruits and vegetable intake was found in 24 of 25 studies for lung cancer. Fruit was significantly protective in cancers of the esophagus, oral cavity, and larynx. In 26 of 30 studies, there was a protective effect of fruit and vegetable intake with respect to cancers of the pancreas and stomach, and in 23 of 38 studies for colorectal and bladder cancers (Block and others 1992).

Health benefits of Fruit Juices

Apple Juice

Phytochemical extracts from fruits have strong antioxidant and antiproliferative effects.

For example, researchers have reported that the total antioxidant activity of phytochemicals in 1g of apples with skin is equivalent to 83.3 µmol vitamin C, which equates to an antioxidant value of 100g (3.5 oz) apples being equivalent to 1500 mg of vitamin C (Liu 2003). In other words, vitamin C in apples contributed only < 0.4% of the total antioxidant activity. Therefore, most of the antioxidant activity in the apple comes from phytochemicals, not vitamin C (Liu 2003).

Apple extracts also contain bioactive compounds that inhibit tumor cell growth in vitro.

Phytochemicals in a 50 mg apple with skin per milliliter (on a wet basis) inhibited tumor cell proliferation by 42%. Phytochemicals in a 50 mg apple without skin per milliliter inhibited

14 tumor cell proliferation by 23%. The apple extracts with skin significantly reduced the tumor cell proliferation when compared with the apple extracts without skin (Liu 2003).

Apples are a significant source of flavonoids in human diets, especially in Europe.

Epidemiological data correlated apple consumption with a reduced lung cancer risk. To initially characterize mechanisms by which apples may prevent cancer, studies using apple formulations derived from different apple tissues have shown antioxidative and antiproliferative activity in vitro. Although no attempt has been made to identify the relevant bioactive component(s) in apples, the observed in vitro effects were attributed to the high concentrations of flavonoids in apple peel and flesh, as well as apple procyanidins or non-flavonoid apple fiber, such as apple pectin (Barth and others 2005).

Apple and wine consumption was found to have an inverse association with death from coronary heart disease in postmenopausal women in study of approximately 35,000 women in

Iowa. Catechin and epicatechin phytochemical compounds found in apples were strongly inversely associated with coronary heart disease death. Nevertheless, catechins found in tea were not associated with coronary heart disease mortality in postmenopausal women. The variation may be because apple catechins are more bioavailable than the catechin gallates commonly found in teas (Boyer and Liu 2004).

Apple consumption is inversely linked with asthma and is also associated with general pulmonary health. According to a study conducted in Australia involving 1600 adults, apple and pear intake was associated with a decreased risk of asthma a decrease in bronchial hypersensitivity (Boyer and Liu 2004). In a previous study conducted by Shaheen and others

(2001) surveying approximately 600 individuals with asthma and 900 individuals without asthma in the United Kingdom, apple intake was associated with less asthma in adults. General fruit and

15 vegetable intake was weakly linked with asthma while apple intake showed a stronger inverse relationship with asthma. Flavonoids in apples may reduce asthma inflammation through antioxidant, antiallergic, and anti-inflammatory properties. Flavonoids are ardent scavengers of nitric oxide, can inhibit histamine release, arachidonic acid metabolism and cytokine production

(Shaheen and others 2001).

In a study conducted by Knekt and others (2002) to determine the association between flavonoid intake and risk of several chronic diseases, apple consumption was inversely associated with occurrence of all cancers combined, lung cancer, asthma, Type II diabetes, thrombotic stroke, total mortality and ischemic heart disease mortality (Knekt and others 2002).

Purple Grape Juice

Flavonoid compounds from grapes have been proven to protect consumers against heart disease by preventing the oxidation of low density lipoproteins, inhibiting blood clotting and promoting vasodilatation (Wilson and others 1998). The flavonoid components of grape products including red wine and purple grape juice inhibit collagen-mediated platelet aggregation.

Flavonoids in red wine and purple grape juice also reduce the susceptibility of low-density lipoprotein cholesterol (LDL-C) to oxidative stress in vitro. In vitro, flavonoid components of red wine and purple grape juice induce endothelium-dependent vasodilation of arterial rings.

Endothelial dysfunction accelerates the development of atherosclerosis or blood clot formation.

Blood clots in a narrowed artery, may lead to a heart attack or stroke (Stein and others 1999).

Platelets are involved in atherosclerotic disease development, and the reduction of platelet activity by medications reduces the incidence and severity of disease. Red wine and grapes contain polyphenolic compounds, including flavonoids, which can reduce platelet aggregation which has been associated with lower rates of cardiovascular disease. A study to

16 evaluate whether commercial grape, orange and grapefruit juices, if taken daily, would reduce in vivo platelet activity was conducted (Keevil and others 2000). A randomized cross-over design was used where 10 healthy human subjects ages 26-58 years, five of each gender drank 5-7.5 ml/kg body weight a day of purple grape juice, orange juice or grapefruit juice for 7-10 days each. Platelet aggregation at baseline was compared to results after consumption of each juice for the test period. According to the results, drinking purple grape juice for one week reduced the whole blood platelet aggregation response to 1mg/L of collagen by 77% (from 17.9 + 2.3,

P=0.0002). Orange juice had no effect on platelet aggregation. The purple grape juice had approximately three times the total polyphenolic concentration of the citrus juices and was a potent platelet inhibitor in healthy subjects, while the citrus juices showed no effect. The platelet

inhibitory effect of the flavonoids in grape juice may decrease the risk of coronary thrombosis

and myocardial infarction (Keevil and others 2000), supporting consumption of this juice for

human health.

Endothelial dysfunction is a critical event in the pathogenesis of atherosclerosis and its

clinical manifestations. This is due to the fact that endothelial function in the human brachial

artery is closely related to endothelial function in the human coronary artery. Purple grape juice

(GJ) enhances endothelial vasodilation by increasing nitrogen oxide production. In a recent study

conducted by Stein and others (1999), incubation of human blood with a 1/1000 dilution of

purple grape juice increased nitrogen oxide release from aggregating platelets by 3-fold (p=0.01).

Since nitrogen oxide interacts with superoxide, release of super oxide by aggregartion platelets

also was measured before and after treatment with purple GJ; a 55% decrease in superoxide

release was observed (p<0.01). In human subjects, who ingested 7 mL of purple GJ for 14 days,

17 significant increases in NO release and decreases in superoxide release were observed (Stein and others 1999).

Pomegranate Juice

Pomegranate fruits are widely consumed fresh and as a beverage, such as pomegranate juice (PJ). Punicalagin, which occurs as isomers, is the predominant ellagitannin (ET) present in

PJ. The potent antioxidant properties of PJ have been attributed to its high content of punicalagin isomers that can reach levels 2 g/L juice. Pomegranate fruit extracts and its purified ellagitannins inhibit the proliferation of human cancer cells and modulate inflammatory subcellular signaling pathways and apoptosis. Pomegranate fruit extract has also been shown to significantly reduce prostate tumor growth and prostate specific antigen levels in athymic nude mice implanted with

CWR22Rv1 prostate cancer cells (Seeram and others 2006a).

There are limited treatment options for prostate cancer patients who have undergone primary therapy with curative intent and who have progressive elevation of their prostate- specific antigen (PSA). A study was conducted in patients with recurrent prostate cancer and have a detectable PSA>0.2 and <5 ng/mL and Gleason score ≤ 7. Patients were treated with 8 ounces of pomegranate juice daily (Wonderful variety, 570 mg total polyphenol gallic acid equivalents) until disease progression. The average doubling time (how long it takes for PSA levels to double, a signal that the cancer is progressing) is about 15 months. The mean PSA doubling time significantly increased with treatment from a mean of 15 months at baseline to 54 months posttreatment (P<0.001). Pretreatment and posttreatment comparison of patient’s serum on the growth of LNCap showed a 12% decrease in cell proliferation and a 17% increase in apoptosis (P= 0.0048 and 0.0004, respectively), a 23% increase in serum nitric oxide (P= 0.0085)

18 and significant (P<0.02) reductions in oxidative state and sensitivity to oxidation of serum lipids after versus before pomegranate juice consumption (Pantuck and others 2006).

Only recently has pomegranate juice and encapsulated pomegranate extract become widely available. In a study conducted to evaluate the antioxidant activity of pomegranate juice in comparison to red wine and a green tea infusion, the antioxidant activity of the experimental pomegranate obtained in the laboratory from pomegranate arils by a hand press was twice those of red wine and green tea. The activity was lower in the experimental juice prepared from frozen arils, showing that during the freezing process, some antioxidant compounds are degraded or transformed. The antioxidant activity of both commercial pomegranate juices was nearly three times that of wine and tea, suggesting that the industrial process to obtain the juices either increased the content of pomegranate antioxidants or enhanced their activity (Gil and others

2000).

A study reported high antioxidant activity of pomegranate juices (Cerd´a and others

2003). This activity was associated with the high content of phenolic compounds that were identified as punicalagin isomers, ellagic acid derivatives, and anthocyanins (delphinidin, cyanidin and pelargonidin3-glucosides and 3, 5-diglucosides). Punicalagin is an ellagitannin in which ellagic acid is linked to a glucose molecule. The punicalagin isomers have been reported to be responsible for the high antioxidant capacity of pomegranate juice. These compounds impart the characteristic red or yellow color of pomegranate husk, and are extracted with the juice during processing (Cerd´a and others 2003).

A study conducted to investigate the capacity of pomegranate juice (PJ) to protect nitric oxide against oxidative destruction found PJ to be a potent inhibitor of superoxide anion- mediated disappearance of nitric oxide. PJ was also found to strengthen the anti-proliferative

19 action of nitric oxide on vascular smooth muscles cell proliferation of rat’s aorta. To determine whether PJ is capable of increasing the production of NO by vascular endothelial cells, PJ was tested for its capacity to upregulate and/or activate endothelial NO synthase (Enos) in bovine pulmonary artery endothelial cells. PJ showed no effects on eNOS protein expression or catalytic activity. In addition, PJ did not enhance promoter activity in the eNOS gene. Therefore, PJ has potent antioxidant activity that results in marked protection of NO against oxidative destruction, thus resulting in strengthening of the biological actions of NO (Ignarro and others 2006).

Pathological induction of matrix metalloproteinase’s (MMPs) plays an important role in the pathogenesis of osteoarthritis (OA) and inflammatory diseases (Ahmed and others 2005).

According to a study conducted at the Case Western Reserve University School of medicine, pomegranate fruit extracts (PFE) possesses anti-inflammatory effects by blocking of enzymes that contribute to osteoarthritis. For example, pretreatment of human OA cartililage explants with

PFE inhibited IL-1β-induced breakdown of the cartilage extracellular matrix. In a similarly treated OA chondrocytes, IL-1β-induced expression of MMPs was also inhibited, indicating that

PFE is a potent inhibitor of cartilage matrix degrading enzymes (Ahmed and others 2005).

Ellagic acid is known to have anti-cancer actions found in substantial amounts in the peels, juice and seed oil of the pomegranate fruit (Lansky and others 2005). Polyphenol-rich fractions of pomegranate fermented juice and peels, and pomegranate seed oil, when combined, supra-additively inhibit proliferation, invasion and secretory phospholipase A2 (Spla2) expression in human prostate cancer cells. To evaluate a possible contribution of this compound, a study was conducted. Ellagic acid was used in a double blind assay for in vitro invasion in which hepatocyte growth factor (HGF) was used to stimulate invasion of 5 x 104 human PC-3

20 prostate cancer cells added to each Matrigel pre-coated chamber of a 24-transwell system

(Lansky and others 2005).

A study conducted to investigate the effects of pomegranate juice on ischemic coronary heart disease, demonstrated that daily consumption of pomegranate juice for three months may decrease myocardial ischemia and improve myocardial perfusion in patients who have ischemic coronary heart disease (CHD) [Sumner and others 2005]. This was illustrated by an average improvement of 17% in myocardial perfusion in the experimental group and an average worsening in myocardial perfusion of 18% in the control group after only three months (Sumner and others 2005).

Cranberry Juice

Laboratory and animal studies have shown that berries have anticancer properties. The biological activities of berries are partially attributed to their high content of a diverse range of phytochemicals which includes tannins such as ellagitannins (Seeram and others 2006b). Berry bioactive impart anticancer effects through various complementary and overlapping mechanisms of action including the induction of metabolizing enzymes, modulation of gene expression and their effects on cell proliferation, apoptosis, and subcellular signaling pathways (Seeram and others 2006b).

The term oxidative stress is applied in vivo to situations in which elevated levels of free radicals or other reactive oxygen species (ROS) can cause either direct or indirect damage to the body. Oxidative stress-related illnesses have been reported to include cancer, cardiovascular disease, Parkinson’s disease and possibly Alzheimer’s disease. Studies have shown that polyphenols may decrease oxidative stress through indirect antioxidant action, such as the inhibition of ROS-producing enzymes like myeloperoxidase, lipoxygenase, cyclooxygenase, and

21 xanthine oxidase [XO] (Dew and others 2005). For example, in a study conducted by Dew and others (2005), cranberry and purple grape juices were found to inhibit XO, with inhibitory concentration of 50% (IC50) values of 2.4 ±0.1 and 3.5 ± 0% (estimated) of original

concentrations, respectively. In the same study, tomato juice was found to mildly inhibit XO,

with an approximate IC50 value of 24 ± 0.55% of original concentration. Apple, carrot, and

pineapple juices were found to promote XO activity slightly in a non-dose-dependent manner.

Orange and pink grapefruit juices were found to promote XO activity, with approximate

promontory concentration of 50% (PC50) values of 7 ± 1 and 9 ± 1% of original concentration,

respectively. The potent inhibitory behavior of cranberries and purple grapes may be derived in

part from anthocyanins, which are responsible for the deep red and blue colors in many plants

(Dew and others 2005).

The flavanoid and hydrocinnamic acid derivatives in cranberry juice reduce the oxidation

of LDL and LDL mobility (Vattem and others 2005). In a study performed by Wilson and others

(1998), cranberry extract containing 1,548 mg gallic acid equivalents/L was used to evaluate

how it affected low density lipoprotein (LDL) oxidation induced by 10 micromolar cupric

sulfates. LDL were incubated in the presence of cupric ions using 0.00%, 0.10%, 0.05%, 0.01%,

and 0.005% dilutions of cranberry extract with a pre-dilution pH of 2.5. Cranberry extract

inhibited thiobarbituric acid reactive substances (TBARS) formation significantly at a 0.10%

dilution, and LDL relative electrophoretic mobility (REM) was significantly increased to a

0.05% dilution (p<0.002) (Wilson and others 1998).

HPLC Analysis

There are two mode of operation in HPLC analysis which includes isocratic and gradient elution.

The isocratic elution is where the mobile phase mixture is prepared to the desired composition

22 manually and stored in a single reservoir, or a solvent proportioning valve is programmed to mix solvents at a constant proportion mixture from the reservoirs to produce the desired mixture composition (Robinson and others 2005). In the gradient elution mode, the solvent proportioning valve is also programmed to mix solvents from the reservoirs to produce the desired mixture composition but in this case, the solvent strength of the mobile phase varies with time by changing the mixture proportions (Robinson and others 2005). For example, in a study conducted by Soong and Barlow (2006), to identify the amount of gallic and ellagic acid in longan seed and mango kernel using a reversed-phase high performance liquid chromatography

(RP-HPLC) coupled with photodiode array detection (DAD), solvent gradients were formed, by the dual pumping system, by varying the proportion of solvent A [water-acetic acid (97: 3, V/V)] to solvent B (methanol). Solvent B was increased to 10% in 10 min and subsequently increased to 70% in 40 min at a flow rate of 0.9ml min-1. The phenolic compounds were detected at 280

and 360 nm (Soong and Barlow 2006). In another study conducted by Seeram and others 2005 to

determine the amount of ellagitannins (ETs) in a pomegranate fruit husk/peel using a HPLC, the

mobile phase solvent A (2% CH3COOH/H2O) and solvent B (2% aqueous CH3COOH/MeOH)

was used under linear gradient conditions starting with 99% A in B for 5 min to 40% A in B over

40 min, hold time, 5 min with a flow rate of 1.0 mL/min. Punicalagin and EA were detected at

378 and 366 nm respectively (Seeram and others 2005).

Sensory Evaluation

Sensory evaluation or sensory analysis is the use of human senses to measure the flavor

and sensory characteristics of foods and other products (Moskowitz 1988). Sensory evaluation of

food is important to many industries that sell consumer goods and food products because the

human senses also integrate their input so that changes in one sense can be perceived as changes

23 in another. For example, a change in smell can sometimes be perceived as a change in taste of a product. In order to successfully sell the product, consumer appeal and target consumer populations should be considered (Moskowitz 1988). Quality is a key product component, and food industries often utilize sensory evaluation techniques to test products. Quality of food is often assessed in terms of three main parameters: color, texture, and flavor. Visual perception of color results from activation of the retina by electromagnetic waves in the visible spectrum.

Flavor characteristics include taste and odor. Taste sensations are produced as substances dissolved in the saliva interact with the taste buds in the papillae on the tongue (Weaver and

Daniel 2003). Quality can be determined using objective or subjective measurements. Objective evaluation involves the use of laboratory instruments with no involvement of the senses.

Subjective evaluation is done by either trained or untrained human observers (OSU 1998).

Total perception of food is a complex experience based upon multiple senses: taste per se, which includes sweet, sour, salty and bitter and thermoreception and nociception, for example, that caused by pungent spices and irritants. Taste proper is commonly divided into four categories of primary stimuli: sweet, sour, salty and bitter. Sweet is evoked by solutions of low concentrations of inorganic salts, sugars, and various nitrogen compounds. Bitter can be associated with hydrophobic amino acids and alkaloids. Salt, sugars and monosodium glutamate can also be used to enhance the flavor (Siegel and others 1999).

Taste Panels

The impact of technology, the use of more complex raw materials, and related developments make it difficult for experts to be as effective in descriptive testing as in the past

(Stone and Sidel 2004). Formal descriptive analysis and the separation of the individual expert from sensory evaluation received its major challenge from the development of the Flavor Profile

24 method. The investigators demonstrated that it was possible to select and train a group of individuals to describe their perception of a product in some agreed sequence, leading to actionable results without dependence on the individual expert (Stone and Sidel 2004).

Test Methods for Taste Panels

Before describing specific test methods, reviewing the fundamental issues on which all descriptive methods are based is important. This includes the participants’ selection process, the extent and duration of the study, the quantification of the panel, and finally method of data analysis. Sensory evaluation must have tools with which to work, including the methods used to evaluate the products, such as difference tests and acceptance/preference tests (Stone and Sidel

2004). While conducting sensory evaluation, the researcher should be familiar with all the different sensory methods to be used in order to apply them properly. Understanding alternative test methods also reduces unwarranted reliance on a single method to test options and solve problems.

Selection of Panelists

Regardless of the source of the participants in a sensory test, they must be qualified to participate. Failure to use appropriately qualified panelists has a significant impact on credibility of the study. There must be a formal program for participants’ selection, so as to improve the level of sensitivity, to match the panel with a specific sensory problem, and to increase confidence in the conclusions derived from the test results (Stone and Sidel 2004). Individuals selected should have normal acuity and perception, above-average interest in odor and flavor work, and the ability to work cooperatively with others in a group setting. These personal qualities are determined by test scores and through a personal interview (Moskowitz 1988).

25 Screening Procedures

Once individuals have indicated a willingness to participate, they are required to participate in a series of screening tests to determine their level of skill. A threshold test is designed to determine the minimum concentration at which a stimulus can be detected as different from a blank (detection threshold) or can be recognized as having a typical taste

Moskowitz 1988). The following are some of the screening tests used to determine the candidates’ taste ability (Moskowitz 1988).

Identification Test- The candidate is given a false suggestion in order to determine their independence of perceptual judgment (Moskowitz 1988).

Basis taste test- Four basic taste solutions (sweet, sour, salty, and bitter), one blank, and one duplicate basis taste. The concentrations are above threshold levels. Confusion between description of sour and bitter taste is often encountered. This test helps to determine whether the candidate displays the confusion of sour and bitter and also if the candidate is able to identify these basic tastes (Moskowitz 1988).

Ranking test- Requires that the candidate perceive different levels of taste in a flavored medium where panelists have to isolate elements from a complex selection (Moskowitz 1988).

Samples should be presented simultaneously, if possible, or else sequentially. The samples should be presented in a balanced randomized order. The task is to rate or rank each sample using the specified scale. The set may be presented once only, or several times with different coding (Meilgaard and others 1987a). The candidate is rated in how he/she applied himself in taking the tests, level of confidence, interest and attitude toward the tests (Moskowitz

1988).

26 CHAPTER III

MATERIALS AND METHODS

Materials

The reference standard of ellagic acid was purchased from Fisher Scientific and was used as received. Pomegranate juice, 100%, (Pomwonderful LLC), 100% purple grape juice (Tropicana

Products, Inc), 100% apple juice (unsweetened) [Wal-mart stores, Inc], and 27 % cranberry juice

(other ingredients include filtered water, high fructose corn syrup, cranberry juice concentrate)

[Tropicana Products Inc] were purchased single strength in plastic bottles from local grocery stores in Bowling Green, Ohio. The juices were stored at room temperature before opening, but were refrigerated at 5° C in covered containers thereafter.

High Pressure Liquid Chromatography-Ultraviolet (HPLC-UV) Analysis of Ellagic Acid

The concentration of ellagic acid for each of the individual juices was determined using high performance liquid chromatography-ultraviolet (HPLC-UV). Analysis was performed using a Hewlett Packard1050 HPLC instrument using a C18 column, 4.6 X 150 mm, 5µm (SGE

Incorporated). The instrument was operated in isocratic mode (mobile phase of constant

composition). The mobile phase was manually prepared by mixing 45% methanol (100% HPLC

grade), 64 % deionized water, and 1% acetic acid (by volume), and all runs were at a flow rate of

1.0 ml/minute. The mobile phase was degassed with helium from a helium tank prior to each

operating session to purge other dissolved gases. Detection was by UV absorbance at 260 nm.

The instrument had a 20 µl syringe loop, so all injected samples were 20 µl.

27 Sample Processing and Analysis

Samples of juice (10 mL) were mixed with equal volumes (10 mL) of 2 Molar hydrochloric acid (HCl). The samples were placed in boiling water bath for 30 min and then allowed to cool for 5 minutes. The samples were each filtered with a 4 mm syringe filter

(0.45µm) [Alltech associates, Inc]. The acid/heat process was used to hydrolyze the ellagotannins to allow the analysis to determine total ellagic acid content of the samples. Some pomegranate samples were mixed with HCl but not heated, to allow measurement of free ellagic acid.

Ellagic acid standard of 166 ppm was prepared in methanol (where EA is soluble), which was then diluted to prepare the other standards. Ellagic acid standards were prepared at dilutions ranging from 10 ppm to 150 ppm in a solvent (methanol) similar to the mobile phase. The standards were not acidified or heated but were injected directly into the HPLC.

Soluble solids content

An Abbe-3L model refractometer (Spectronic Instruments, Inc) was used to determine the amount of soluble solids content in each of the juices (°Brix values). This was to ensure that the juice blends were within the °Brix values specifications (Table 1).

Sensory Evaluation of the Juices

The juices were combined (Figure 2) at the following ratios 1:1, 1:3, 3:1, (v/v). Sensory evaluations were conducted for each mixture to determine the most acceptable mixtures by the panelists. Hedonic rating and ranking test were used to determine the panel’s overall acceptability of the combined juices. The hedonic rating had a scale of 1-9 (1- dislike extremely and 9-like extremely). The ranking test had a scale of 1-6 (1- least preferred and 6-most preferred). The color, sweetness and mouthfeel of the juice blends were each tested separately.

28 Table 1- °Brix values for individual and combined juices Type of juice U.S Average º Brix º Brix Apple (A)a 13.3e 12.7 Purple grape (G)b 21.5e 17.3 Pomegranate (P)c 18.1e 16.9 Cranberry (C)d 10.5e 14.6 0.5 combination GC 15.3 GA 14.4 GP 16.7 PC 15.2 CA 13.1 PA 14.2 0.25 combination GC 14.7 GA 13.2 GP 16.5 PC 14.7 CA 12.9 PA 13.3 0.75 combination GC 16.1 GA 15.6 GP 16.4 PC 16.2 CA 13.6 PA 15.3 aApple, bPurple grape juice, cPomegranate, dCranberry e(Source: http://www.honeycreek.us/brix.htm)

G P C A

GC GA GP PC CA PA

Figure 2: Flowchart of juice combination for sensory evaluation

Key: A- Apple G- Purple grape juice C- Cranberry P- Pomegranate

Taste Panelists

With the approval of the Bowling Green State University Human Subjects Review Board

(HSRB Project No: H07T141GE7), 10 Bowling Green State University students were recruited through word of mouth, emails, and random selection at the Bowen-Thompson Student Union building to serve as taste panelists. The participants were 20 to 45 years of age. The participants were briefed on the thesis project and given a pre-screening questionnaire to complete (Appendix

A). The participants also had to sign a consent form allowing them to voluntarily participate and withdraw as they wish prior to or during the testing (Appendix B).

The individuals participated in a discrimination and matching test (Appendix C). The discrimination test was to help determine the ability of each candidate to detect the differences in samples while the matching test helped to determine a candidate’s ability to discriminate differences among several stimuli presented at intensities well above threshold level (Meilgaard and others 1987b). Participants qualified based on the results of their sensory screening test and their willingness to participate in the project. Those who scored less than 75% in the sensory

30 screening test were disqualified. Those individuals who completed the study were given a $10 gift certificate for their participation.

Table 2- Concentrations of the matching test taste solutions Flavor Concentration g/L Concentration g/L Sweet-sucrosea 20 DWe Acid (sour)-citric acidb 0.5 0.5b / 0.25c / DWe Bitter-quinine sulfatec 0.5 .25 c / 20a / DWe Salty-sodium chlorided 2 .25 c / 20a / 2d / DWe a Sweet-sucrose c Quinine sulfate e Distilled water b Citric acid d Sodium chloride

Table 3-Concentrations of the discrimination test taste solution Sample Concentration g/L Sample Distilled water Sucrose solution 7.0 Distilled water Distilled water Sucrose solution 14.0 Distilled water Distilled water Sucrose solution 28.0 Distilled water Distilled water Sucrose solution 56.0 Distilled water

Participants’ Screening Procedures

Once individuals had indicated a willingness to participate, they were required to

participate in a series of screening tests to determine their level of tasting skill. This screening

process had two stages, the first was completion of a product attitude survey, and second, participation in a series of selected sensory tests (Stone and Sidel 2004). People are variably sensitive to compounds provoking the four basic tastes; sweet, sour, bitter and salty. The lowest concentration that can be recognized as one of the basic tastes is known as the recognition

threshold (Weaver and Daniel 2003). Taste threshold sensitivity, taste threshold recognition, and

primary taste identification were conducted consecutively. The order of testing was to prevent

the potential taste panelists from exposure to the four taste modalities (used in the primary taste

identification) prior to their sensitivity and recognition testing. Those who had no known

31 allergies to juices, and who demonstrated a minimum level of sensitivity and reliability qualified to be in the sensory panel (Stone and Sidel 2004).

In order to select taste panelists with normal taste perception, sensory screening tests were conducted (Appendix B). The individuals participated in a matching test to help determine a candidate’s ability to discriminate differences among several stimuli presented at intensities well above threshold level (Meilgaard and others 1987b). The individuals were presented with four coded but unidentified products for familiarization purposes. The participants were then presented with a randomly numbered set of eight samples, of which a subset was identical to the initial set. The participants were asked to identify the similar samples (Meilgaard and others

1987b).

The individuals then participated in triangle taste threshold tests. This helped determine the candidate’s ability to detect differences among similar products with ingredient variables

(Meilgaard and others 1987b). The design for the triangle, taste threshold test for the potential taste panelists was as follows: Each individual was presented four different groupings of three solutions. Two of the solutions in each group were distilled water and the other a sucrose solution. The concentration of the sucrose solution was increased with each grouping. The potential taste panelist were asked to select the different sample in each grouping and to continue through each successive grouping until the taste of the different sample could be identified as one of the four basic tastes that is sweet, salty, sour, or bitter (Meilgaard and others 1987a).

Finally, the individuals were required to taste four solutions, representing each of the basic tastes (sucrose- sweet, sodium chloride- salty, citric acid- sour, quinine sulfate- bitter).

They had to identify each correctly to qualify for the taste panel (Meilgaard and others 1987b).

32 Tasting Location

A Food and Nutrition laboratory was the tasting location. Temporary booths made from white poster boards were used to discourage any visual distraction for the panelists. The tasting booths were placed on separate tables in the Food and Nutrition laboratory and adequately lighted. Noise and odor was eliminated from the tasting environment. The samples were presented in small 1oz plastic cups.

Tasting Procedure

The juices were combined (Figure 3) at the following ratios 1:1, 1:3, 3:1, (v/v) for each combination.

G P C A

GC GA GP PC CA PA

Figure 3: Flowchart of juice combination for sensory evaluation Key: A- Apple G- Purple grape juice C- Cranberry P- Pomegranate

The samples were presented in small plastic cups. The juice samples were assigned three digit random codes for identification. The juices were served at room temperature (20ºC) because taste sensitivity is greatest at 20-30ºC. Twenty five milliliters of juice was poured into coded, one ounce, small plastic cups just prior to sensory evaluation. The tests were conducted in duplicate.

33 In this study, a ranking and hedonic rating scale test were used to determine which sample was mostly preferred and the overall acceptance for color, sweetness, mouthfeel by the panelists (Appendix D). In a 1-6 point ranking; where most preferred was 6 and least preferred was 1, the juices were grouped into three categories with six different combinations in each category based on their concentration; 1:1, 1:3, and 3:1. The panelists also showed their overall acceptance of color, sweetness and mouthfeel of the juice samples on a 1-9 hedonic rating scale;

9- like extremely, 8- like very much, 7- like moderately, 6-like slightly, 5- neither like or dislike,

4-dislike slightly, 3-dislike moderately, 2- dislike very much, 1-dislike extremely (Appendix D).

The taste panel was composed of seven females and four males with their ages ranging between 20 to 35 years. None of the panelists smoked, while most of them indicated they drink beer.

Statistical Testing of Collected Data

Descriptive statistics, such as median, standard deviation and range, were provided for all variables before conducting the inferential tests. Repeated measures analysis of variance

(ANOVA) was used to analyze the ratings of the juices overall acceptability. Friedman test was used to analyze the rankings of the panelists’ preferences of the juices.

34 CHAPTER IV

RESULTS AND DISCUSSION

Four commercially available juices were combined into 18 different combinations as shown in Table 4. The combined juices were tested for preference and overall acceptance based palatability characteristics of color, mouthfeel and sweetness. The individual and combined juices were then analyzed for the amount of EA using a HPLC–UV (Table 6). The amount of ellagic acid in each juice was determined using a standard calibration curve (Figure 16). A regression equation for the line was y=1.5895x (where x is the concentration of EA; y is the area under the curve).

Selection of taste panelists

To select potential taste panelists, taste threshold sensitivity, taste threshold recognition, and primary taste identification were conducted consecutively. The scores of the potential taste panelists on these tests are given in Table 7 (Appendix C). The order of testing was to prevent the potential taste panelists from exposure to the four taste modalities (used in the primary taste identification) prior to their sensitivity and recognition testing. Tasting the sample in the previously mentioned order helped in the identification of potential taste panelists who had the most sensitive taste recognition threshold levels. Those panelists who were able to sense the odd samples, and in addition had an early and correct recognition of the taste of the odd samples, qualified as panelists with the most acute sense of taste. Nevertheless, those potential panelists able to match correctly the four basic tastes were also considered qualified taste panelists.

Seventeen candidates were recruited for the study, but only 16 qualified as taste panelists. Five candidates did not attend all of the tasting sessions and therefore, were disqualified from the study. Eleven candidates successfully completed all the sessions of this study.

35 Sensory Testing of Juice Mixtures

10.00

Grape and Cranberry

8.00 Grape and Apple

Grape and Pomegranate 6.00

Pomegranate and Cranberry

4.00 Cranberry and Apple Mean sweetmess

2.00 Pomegranate and Apple

0.00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Juice combination

Figure 4- The mean of a hedonic scale test (1- 9 hedonic rating scale; 9- like extremely, 1- dislike extremely) for overall acceptance of sweetness of 18 different combinations of juices (Error bars: 95% CI). The mean sweetness is the sum of the rating of each combination divided among 11 panelists. The juices were combined at the following ratios: 1-6 (1:1), 7-12 (1:3), 13- 18 (3:1).

A repeated measures analysis of variance was conducted to assess differences in overall

acceptance of sweetness based on 18 juice blends. The results of the overall analysis were

significant, F (17,170) = 6.99, P<.0001. The rating scale used was 1- 9; 9-like extremely and 1-

dislike extremely. Figure 4 illustrates that 1:3 purple grape-apple combination (8) had the most

overall acceptance of sweetness (7.68 ± 0.78) followed by 3:1 purple grape-apple (14)

combination. Mixtures 8 and 14 were perceived as significantly better than mixtures 16, 18, 6, 4,

9, 10. Mixture 11 was identified as significantly better than 16, 18, and 6, while mixtures 13, 2,

15, 5 were reported as better than 16 and 18. Also, combinations 17, 12, 1, 7, and 3 were

perceived as significantly better than 16. Therefore, 3:1 pomegranate-cranberry mixture (16) had

36 the least overall acceptability (4.18 ± 1.70) followed by the 3:1 pomegranate-apple juice combination (18).

10.00 Grape and Cranberry

Grape and Apple 8.00

Grape and Pomegranate

r 6.00 Pomegranate and Cranberry Mean colo 4.00 Cranberry and Apple

Pomegranate and 2.00 Apple

0.00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Juice combination

Figure 5 - The mean of a hedonic scale test (1- 9 hedonic rating scale; 9- like extremely, 1- dislike extremely) for overall acceptance of color of 18 different combinations of juices (Error bars: 95% CI). The mean color is the sum of the rating of each combination divided among 11 panelists. The juices were combined at the following ratios: 1-6 (1:1), 7-12 (1:3), 13-18 (3:1).

A repeated measures analysis of variance was conducted to assess differences in overall

acceptance of color based on 18 juice blends. The results of the overall analysis were significant,

F (17,170) =3.05, P<.0001. The rating scale used was 1- 9; 9-like extremely and 1-dislike

extremely. The 1:1 purple grape-cranberry mixture (1) had the highest mean score (7.27± 1.14),

and therefore, had the most overall acceptance of color (Figure 5). Mixture 1 was rated

significantly better than mixtures 12, 11, and 8. However, mixtures 7, 14, 17 and 10 were all

37 perceived to be significantly better than 12. Consequently, 1:3 pomegranate-apple mixture (12) had the lowest mean score, (4.59 ± 1.28) and thus, had the least overall acceptability of color.

10.00

Grape and Cranberry

8.00 Grape and Apple

Grape and Pomegranate

6.00 Pomegranate and Cranberry

4.00 Cranberry and Apple Mean mouththfeel Pomegranate and Apple 2.00

0.00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Juice combination

Figure 6- The mean of a hedonic scale test (1- 9 hedonic rating scale; 9- like extremely, 1- dislike extremely) for overall acceptance of mouthfeel of 18 different combinations of juices (Error bars: 95% CI). The mean mouthfeel is the sum of the rating of each combination divided among 11 panelists. The juices were combined at the following ratios: 1-6 (1:1), 7-12 (1:3), 13- 18 (3:1).

A repeated measures analysis of variance was conducted to assess differences in overall

acceptance of mouthfeel based on eighteen juice blends. The results of the overall analysis were

significant, F (17,170) =10.08, P<.0001. The rating scale used was 1- 9; 9-like extremely and 1-

dislike extremely. Sample 14 (3:1 purple grape-apple blend) had the highest mean score of

overall acceptance of mouthfeel (7.54 ± 0.85) [Figure 6]. There was no significant difference

between mixture 14 and 8. Nevertheless, mixture 14 and 8 was identified as possessing

38 significantly better mouthfeel than mixtures 16, 4, 9, 18, 6, 10, and 3. Also, mixture 15 and 2 was reported to have significantly better mouthfeel than 16, 4, 9, 18, 6, and 10 with an exception of mixture 3. The 1:1 cranberry-apple mixture (5), 11, 13, 12, and 1 was perceived to have significantly better mouthfeel than mixtures16, 4, 9, and 18 excluding mixtures 6 and 10. The 3:1 pomegranate-cranberry mixture (16) had the lowest mean score (4.41 ± 1.99) and therefore, the least overall acceptability of mouthfeel.

Nine separate Friedman tests were conducted to identify differences in the panelists’ rankings of the 18 different juice combinations (6 colors, 6 sweetness, and 6 mouthfeel). The grand medians are indicated in Table 4. As indicated by the results of the nine analyses, only 1:1 color, and 3:1 color were not statistically significant.

Table 4- Friedman test of the panelists’ rankings of color, sweetness, and mouthfeel. Sample Grand median df PR >F 1:1 Color 3.33 5 0.353 1:3 Color 3.54 5 0.022 3:1 Color 3.33 5 0.605 1:1 Sweetness 3.58 5 0.004 1:3 Sweetness 3.54 5 0.024 3:1 Sweetness 3.42 5 0.001 1:1 Mouthfeel 3.54 5 0.001 1:3 Mouthfeel 3.50 5 0.008 3:1 Mouthfeel 3.54 5 0.001

There was no significant difference among the six 1:1 juice combinations for color

rankings. The rankings were not significant at (p <0.05). The ranking scale used was 1-6; 6-most

preferred and 1-least preferred. The 1:1 purple grape-cranberry mixture had the highest mean

score in color preference (4.36 ± 0.97) and therefore, ranked the highest in color preference

followed by 1:1 pomegranate-cranberry mixture (Figure 7). However, 1:1 pomegranate-apple

combination had the lowest mean score (2.77 ± 1.26), and thus this sample ranked the lowest in

color preference.

5.00

GC-Grape and Cranberry

GC GA-Grape and Apple 4.00 PC GP-Grape and Pomegranate

PC-Pomegranate and CA GA GP Cranberry 3.00 PA CA-Cranberry and Apple

PA-Pomegranate and Apple 2.00 Mean color

1.00

0.00 1.00 2.00 3.00 4.0 5.0 6.00 0 0 Juice combination Figure 7 - The mean of a ranking test (1- 6 ranking scale; 6-most preferred and 1-least preferred) for color preference of six different 1:1 combination of juices. The mean color is the sum of the 1:1 combination color ranks divided among 11 panelists.

There was no significant difference among the six 1:1 juice combinations for color rankings. The ranking scale used was 1- 6; 6-most preferred and 1-least preferred. The rankings were not significant at (p <0.05). The 1:1 purple grape-cranberry mixture had the highest mean score in color preference (4.36 ± 0.97) and therefore, ranked the highest in color preference followed by 1:1 pomegranate-cranberry mixture (Figure 7). However, 1:1 pomegranate-apple combination had the lowest mean score (2.77 ± 1.26), and thus this sample ranked the lowest in color preference.

5.00 GC-Grape and Cranberry GC GA-Grape and Apple

4.00 PC GP-Grape and Pomegranate GP PC-Pomegranate and

r Cranberry 3.00 GA

CA CA-Cranberry and Apple

Mean colo 2.00 PA-Pomegranate and Apple

1.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 Juice combination

Figure 8 – The mean of a ranking test (1- 6 ranking scale; 6-most preferred and 1-least preferred) for color preference of six different 1:3 combination of juices. The mean color is the sum of the 1:3 combination color ranks divided among 11 panelists.

There was an overall significance in color preference rankings at (p< 0.05). The ranking scale used was 1- 6; 6-most preferred and 1-least preferred. The 1:3 purple grape-cranberry mixture (1) had the highest mean score in color preference (4.82 ± 1.10) and therefore, ranked the highest in color preference followed by sample 4 with a mean score of (4.18 ± 1.08) [Figure

8]. There were no significant differences in color among samples 1:3 purple grape and cranberry juice, 1:3 pomegranate and cranberry and 1:3 purple grape-pomegranate, but there was a significant difference in color between the three samples and samples 1:3 pomegranate-apple juice, 1:3 cranberry-apple juice, and 1:3 purple grape-apple juice. The 1:3 grape-apple juice and

1:3 grape-pomegranate had higher rankings in color preference than 1:3 cranberry-apple juice and pomegranate-apple juice. The 1:3 pomegranate-apple mixture had the lowest score (2.41 ±

0.71), and thus ranked the lowest in color preference.

5.00

GC-Grape and Cranberry

GC GA-Grape and Apple 4.00

GP-Grape and Pomegranate GP CA GA PC PC-Pomegranate and Cranberry 3.00

PA CA-Cranberry and Apple

PA-Pomegranate and Apple 2.00 Mean color

1.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 Juice combination

Figure 9 - The mean of a ranking test (1- 6 ranking scale; 6-most preferred and 1-least preferred) for color preference of six different 3:1 combination of juices. The mean color is the sum of the 3:1 combination color ranks divided among 11 panelists.

There were no overall significant differences in the ranking of color among the six 3:1 juice combinations at (p< 0.05). The ranking scale used was 1- 6; 6-most preferred and 1-least preferred. The 3:1 purple grape-cranberry mixture had the highest mean score in color preference

(4.32 ± 0.95), and therefore, ranked the highest in color preference followed by 3:1 purple grape- pomegranate mixture (Figure 9). However, 3:1 pomegranate-apple combination had the lowest mean score (2.86 ±1.53), and thus ranked the lowest in the 3:1 juice combination color preference.

5.00 GC-Grape and Cranberry GA GA-Grape and Apple GC 4.00 CA GP-Grape and Pomegranate

PC-Pomegranate and Cranberry

3.00 GP CA-Cranberry and Apple

PA PA-Pomegranate and Apple

2.00 PC Mean sweetness

1.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 Juice combination

Figure 10 - The mean of a ranking test (1- 6 ranking scale; 6-most preferred and 1-least preferred) for sweetness preference of six different 1:1 combination of juices. The mean sweetness is the sum of the 1:1 combination sweetness ranks divided among 11 panelists.

There was an overall significance in sweetness preference ranking of the six different 1:1 combinations of juice at (p< 0.05). The ranking scale used was 1- 6; 6-most preferred and 1-least preferred. The 1:1 purple grape-apple mixture had the highest mean score in sweetness preference (4.77 ± 1.12), and therefore, ranked the highest in sweetness preference (Figure 10), followed by 1:1 grape-cranberry juice with a mean score of (4.32 ±1.36). There were no significant differences in sweetness among 1:1 grape-pomegranate juice, grape-cranberry juice and grape-apple juice, but there was a significant difference in sweetness between the previously mentioned three samples and 1:1 pomegranate-cranberry juice, 1:1 pomegranate-apple juice, and

1:1 grape-pomegranate mixtures. The 1:1 grape-pomegranate had a higher sweetness preference than 1:1 pomegranate-cranberry juice and 1:1 pomegranate-apple juice combinations. The 1:1

43 pomegranate-cranberry mixture had the lowest score in sweetness preference (2.09 ± 0.81), and thus ranked the lowest in sweetness preference.

5.00 GC-Grape and Cranberry

GA-Grape and Apple GA 4.00 CA GC GP-Grape and Pomegranate PA PC-Pomegranate and Cranberry

3.00 CA-Cranberry and Apple

PC PA-Pomegranate and Apple GP 2.00 Mean sweetness

1.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 Juice combination

Figure 11 - The mean of a ranking test (1- 6 ranking scale; 6-most preferred and 1-least preferred) for sweetness preference of six different 1:3 combination of juices. The mean sweetness is the sum of the 1:3 combination sweetness ranks divided among 11 panelists.

There was an overall significance in sweetness preference ranking of the six different 1:3 combinations of juice at p< 0.05. The ranking scale used was 1 6; 6-most preferred and 1-least preferred. The 1:3 purple grape-apple mixture had the highest mean score in sweetness preference (4.36 ± 1.12), and therefore, ranked the highest in sweetness preference followed by

1:3 cranberry and apple juice combinations with a mean score of (4.23 ± 1.67) [Figure 11]. There were no significant differences in sweetness among samples 1:3 grape-apple juice, 1:3 cranberry- apple juice, 1:3 grape-cranberry and 1:3 pomegranate and apple juice combinations, but there was a significant difference in sweetness between the previously mentioned four samples and 1:3 grape- pomegranate juice and 1:3pomegranate and cranberry juice combinations. The 1:3

44 pomegranate-apple juice had a higher sweetness preference than 1:3 pomegranate-cranberry juice and 1:3 grape-pomegranates. The 1:3 purple grape-pomegranate mixture had the lowest mean score (2.32 ±1.60) and thus ranked the lowest in sweetness preference.

5.00 GC-Grape and Cranberry GC GA

GA-Grape and Apple

4.00 GP GP-Grape and Pomegranate CA PC-Pomegranate and Cranberry 3.00

CA-Cranberry and Apple

2.00

PC Mean sweetness PA

1.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 Juice combination

Figure 12 - The mean of a ranking test (1- 6 ranking scale; 6-most preferred and 1-least preferred) for sweetness preference of six different 3:1 combinations of juice. The mean sweetness is the sum of the 3:1 combination sweetness ranks divided among 11 panelists.

There was an overall significance in sweetness preference ranking of the six different 3:1 combinations of juice at (p< 0.05). The ranking scale used was 1- 6; 6-most preferred and 1-least preferred. The 3:1 purple grape-apple mixture had the highest mean score in sweetness preference (4.91 ± 0.80), and therefore, ranked the highest in sweetness preference followed closely by 3:1 grape-cranberry juice combination with a mean score of (4.86 ± 0.87) [Figure 12].

There were no significant differences in sweetness among 3:1 grape-apple juice, 3:1 grape- cranberry, and 3:1 grape-pomegranate, but there was a significant difference in sweetness between the previously mentioned three samples and the following 3:1 combination, cranberry-

45 apple juice, pomegranate-cranberry juice, and pomegranate-apple juice. The 3:1 cranberry-apple juice and 3:1 grape-pomegranate had higher sweetness preference than 3:1 pomegranate- cranberry and 3:1 pomegranate-apple juice mixtures. There was no significant difference in sweetness for 3:1 pomegranate-apple juice, and 3:1 pomegranate-cranberry juice blend. The 3:1 pomegranate-apple mixture had the lowest score in sweetness preference (1.5 ± 0.56) and thus ranked the lowest in sweetness preference.

5.00

GA GC-Grape and Cranberry

GC 4.00 GA-Grape and Apple CA GP-Grape and Pomegranate

PC-Pomegranate and Cranberry 3.00 GP

CA-Cranberry and Apple PA PA-Pomegranate and Apple 2.00 PC Mean mouthfeel

1.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 Juice combination

Figure 13- The mean of a ranking test (1- 6 ranking scale; 6-most preferred and 1-least preferred) for mouthfeel preference of six different 1:1 combinations of juice. The mean mouthfeel is the sum of the 1:1 combination mouthfeel ranks divided among 11 panelists.

There was an overall significance in mouthfeel preference ranking of the six different 3:1 combinations of juice at (p< 0.05). The ranking scale used was 1- 6; 6-most preferred and 1-least preferred. The 1:1 purple grape-apple mixture had the highest mean score in mouthfeel preference (5.23 ± 1.02), and therefore, ranked the highest in mouthfeel preference followed by

1:1 grape-cranberry juice combination with a mean score of (4.41 ± 0.90) [Figure 13]. There

46 were no significant differences in sweetness among 1:1 grape-cranberry juice and 1:1 grape and apple juice mixtures, but there was a perceived significant difference in sweetness between the previously mentioned two samples and the 1:1 cranberry-apple juice, 1:1 pomegranate-apple juice, 1:1 grape-pomegranate, and 1:1 pomegranate-cranberry juice combination. The 1:1 cranberry-apple and 1:1 grape-cranberry and 1:1 pomegranate-cranberry juice blends had higher sweetness preference than 1:1 pomegranate-cranberry juice, 1:1 grape-pomegranate mixtures, and 1:1 pomegranate-apple juice mixtures. The 1:1 pomegranate-cranberry mixture had the lowest score in mouthfeel preference (1.68 ± 0.64) and thus ranked the lowest in mouthfeel preference

5.00

GA GC-Grape and Cranberry GC 4.00 GA-Grape and Apple CA PA GP-Grape and Pomegranate

3.00 PC-Pomegranate and Cranberry PC CA-Cranberry and Apple

Mean mouthfeel 2.00 PA-Pomegranate and Apple GP

1.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 Juice combination

Figure 14 - The mean of a ranking test (1- 6 ranking scale; 6-most preferred and 1-least preferred) for mouthfeel preference of six different 1:3 combinations of juice. The mean mouthfeel is the sum of the 1:3 combination mouthfeel ranks divided among 11 panelists.

There was an overall significance in mouthfeel preference ranking of the six different 1:3 combinations of juice at p< 0.05. The ranking scale used was 1- 6; 6-most preferred and 1-least

47 preferred. The 1:3 purple grape-apple mixture (2) had the highest mean score in mouthfeel preference (4.55 ±1.69), and therefore, ranked the highest in mouthfeel preference followed closely by 1:3 grape-cranberry juice mixture with a mean score of (4.36 ± 1.50) [Figure 14].

There were no reported significant differences in mouthfeel among 1:3 pomegranate-apple juice,

1:3 cranberry-apple juice, 1:3 grape-cranberry juice, and 1:3 grape-apple, but there was a significant difference in sweetness between the previously mentioned four samples and 1:3 grape-pomegranate and 1:3 pomegranate-cranberry juice. The 1:3 pomegranate-apple juice and

1:3 cranberry-apple juice had higher mouthfeel preference than 1:3 pomegranate-cranberry juice and 1:3 pomegranate-apple juice combinations. There was no perceived significant difference in mouthfeel for 1:3 grape-pomegranate and 1:3 pomegranate-cranberry juice. The 1:3 purple grape-pomegranate mixture had the lowest score (1.90 ±1.44) and thus ranked the lowest in mouthfeel preference.

There was an overall significance in mouthfeel preference ranking of the six different 3:1 combinations of juice at p< 0.05. The ranking scale used was 1- 6; 6-most preferred and 1-least preferred. The 3:1 purple grape-cranberry mixture had the highest mean score in mouthfeel preference (5.23 ± 0.59), and therefore, ranked the highest in mouthfeel preference followed closely by 3:1 grape-apple juice mixture with a mean score of (5.18 ± 0.63) [Figure 15]. There were no perceived significant differences in mouthfeel between 3:1 grape-apple juice and 3:1 grape-cranberry juice mixtures, but there was a reported significant difference in mouthfeel between the previously mentioned two samples and 3:1 grape-pomegranate juice, 3:1 cranberry- apple juice, 3:1 pomegranate-cranberry juice and 3:1 pomegranate-apple juice mixtures. The 3:1 grape-pomegranate had higher mouthfeel preference than 3:1 cranberry-apple juice, 3:1 pomegranate-cranberry juice and 3:1 pomegranate-apple juice blends. Although there was no

48 significant difference in mouthfeel between 3:1 pomegranate-cranberry juice and 3:1 pomegranate-apple juice, they both had the lowest mean score in mouthfeel preference (1.82 ±

0.88 and 1.77 ± 0.70) consecutively. However, 3:1 pomegranate-apple mixture had the lowest score and thus ranked the lowest in mouthfeel preference

6.00

GC-Grape and Cranberry

5.00 GC GA GA-Grape and Apple

GP-Grape and Pomegranate 4.00 GP PC-Pomegranate and Cranberry

CA-Cranberry and Apple 3.00 CA PA-Pomegranate and Apple

Mean mouthfeel 2.00 PC PA

1.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 Juice combination

Figure 15 - The mean of a ranking test (1- 6 ranking scale; 6-most preferred and 1-least preferred) for mouthfeel preference of six different 3:1 combinations of juice. The mean mouthfeel is the sum of the 3:1 combination mouthfeel ranks divided among 11 panelists.

HPLC-UV Analysis

Typical HPLC Results

In a typical run, an ellagic acid standard (EA) shows a well defined peak at 8 min and a small impurity peak at about 5 minutes (Figure 16). A typical pomegranate juice sample showed many peaks in the 1- 6 minutes range and an isolated peak in the 8 minute region; this peak was assigned to ellagic acid (Figure 16).

49 Computer software recorded the chromatogram and allowed integration of the EA peak.

The areas were corrected to the detector range selected units and had units of absorbance unit,

(AU) and time (in tenths of second). An EA standard calibration curve was developed (Figure

17). The EA concentration of the juice samples was determined from the peak area by using the equation for linear regression obtained from the standard calibration curve (Table 5). The regression equation for the line was y=1.5895x (where x is the concentration of EA; y is the area under the curve). The results of the juices are an average of duplicates runs for each juice or juice mixtures on (Table 9) [Appendix E].

1.20

EA 30 ppm 1.00 standard

Pomegranate 0.80 hydrolyzed

0.60

0.40 Absorbance (AU)

0.20

0.00 0 1 2 3 4 5 6 7 8 9 10 Time (min)

` Figure 16- HPLC chromatogram of ellagic acid from the ellagic acid standard and pomegranate juice.

y = 1.5895x Figure 17- Ellagic acid standards calibration curve 300

250 200 Area 150 100 50 0 0 50 100 150 200 Ellagic acid (mg/L)

Figure 17-Ellagic acid standards calibration curve

The results in Table 5 show relatively high levels of free EA in pomegranate juice

(approximately 32 mg/L), and the level increased about three fold (103 mg/L) in hydrolyzed pomegranate juice (free + bound EA content).

The results in Table 5 show relatively high levels of free EA in pomegranate juice

(approximately 32 mg/L), and the level increased about three fold (103 mg/L) in hydrolyzed pomegranate juice (free + bound EA content). However, the result failed to confirm the first hypothesis which stated that “A combination of pomegranate and cranberry juice will have a higher concentration of ellagic acid than each juice alone”. This is because hydrolyzed cranberry

(C), apple (A) and purple grape juice (G) samples had EA concentrations ranging between 1- 2 mg/L. Juice mixtures were also analyzed, and results are shown in Table 5. One would hypothesize that the EA of a mixture is simply the weighted average of the EA of the mixtures.

For example, (see the below calculation)

0.25 GP: 1/4 (1.10) +3/4 (103) = 77.5 mg/L while the analytical value is 77.6 mg/L

51 The results of the EA standard used for the calibration graph plotting and sample containing pomegranate were obtained during a session where EA retention time was 8 minutes. Some of the other juices in the results were measured when retention time was about 11 minutes, but these had very low concentration of EA and therefore, including them in the results introduced no uncertainty. The possible causes for the variations in retention time (RT) are:

1. Change in temperature of the column: Although the laboratory room where the

analyses were conducted would have a change in temperature, the change was not

thought to be significant enough to change the column conditions. Therefore,

temperature change was ruled out as a cause of variation.

2. Change in the mobile phase: This should not have been a cause since the same

container of premixed mobile phase was used for most runs, and yet the runs had

RT variation.

3. Change in flow rate caused by valve leaking or pump problem: The amount of

liquid coming from the HPLC detector was measured using a 10 ml volumetric

flask to ensure that the pump was delivering 1mL/min. The liquid collected was

found to reach the 10 mL, and the pump was found to be delivering the required

amount of liquid. It is possible that the mixing valve was operating inconsistently

and adding some liquid from the other storage reservoirs.

Nevertheless, the cause of variation in RT of the EA peaks was not found, and therefore,

the ellagic acid concentrations in the individual juices and the blends had varying retention

time.

52 Table 5- Concentration of ellagic acid in the individual juices and juice blends. Sample Area (AU) Concentration (mg/L) Hydrolyzed pomegranate (P) 163.89 ± 6.60 103.0 Unhydrolyzed pomegranate (P) 50.10 ± 8.30 32.0 Cranberry (C) 3.04 ± 0.57 1.9 Apple (A) 3.03 ± 0.39 1.9 Purple Grape (G) 1.75 ± 0.25 1.1 1 :1 Combination GC 1.26 ± 0.26 0.8 GA 0.80 ± 0.05 0.5 GP 90.26 ± 3.26 56.9 PC 83.21 ± 4.33 52.4 CA 2.77 ± 0.78 1.8 PA 89.63 ± 1.29 56.5 1:3 Combination GC 1.89 ± 0.71 1.2 GA 3.33 ± 0.11 2.1 GP 123.17± 1.16 77.6 PC 58.24 ± 1.25 36.7 CA 1.91 ± 0.25 1.2 PA 54.25 ± 0.11 34.2 3:1Combination GC 0.94 ± 0.10 0.6 GA 1.49 ± 0.05 0.9 GP 46.53 ± 0.06 29.3 PC 153.91 ±11.35 97.0 CA 1.98 ± 0.15 1.2 PA 151.80 ± 2.58 95.6

Another issue was the precipitation of the EA in standards that were 20 ppm and above

50% methanol/ water; this occurred approximately 5 hours after preparation. This required fresh

preparation of standards for runs conducted after 5 hours, or the following day. Nevertheless,

when standards with concentrations of 30-80 ppm were injected to the HPLC, they showed a

linear dependence on the EA concentration, which suggested that when fresh solvent in the

53 HPLC comes into contact with precipitated samples, the precipitate is dissolved into solution again.

The HPLC analysis of juices and standards produced a single, well resolved peak corresponding to ellagic acid. The peaks were broader than anticipated, corresponding to N

(number of theoretical plates) around 5000. To eliminate the possibility that the peak for juices included an additional component, samples were collected after leaving the HPLC detector.

These samples were then analyzed by absorption spectrophotometry and by fluorescence spectroscopy. The fluorescence studies were conducted in samples in water, in methanol and in a pH 9 buffer of sodium borate. Although samples were dilute, the spectra generally matched those of ellagic acid under similar circumstances. The fluorescence of ellagic acid is enhanced and the spectrum shifts when the solute is a borate buffer (spectra diagram not provided).

Most reported ellagic acid and ellagotannins HPLC analysis used gradient methods, while in this study an isocratic mode was used due to the fact that the instrument initially available for the experiment did not support the gradient mode. The mobile phase was 45% methanol, 1% acetic acid and 64% deionized water. The acetic acid was used to help produce sharper peaks, presumably by preventing the deprotonation of the ellagic acid.

In pomegranate juice, the largest fraction of ellagic acid is bound with tannins. The ellagotannins were hydrolyzed (to ellagic acid) by heating in 1M hydrochloric acid for 30 minutes. Tests showed that longer heating did not produce additional hydrolysis product. The total ellagic acid in the hydrolyzed sample is reported. Physiologically, the ellagotannins are hydrolyzed to ellagic acid so the total is the appropriate quantity. Other individual and combined juices used in this study were also hydrolyzed for 30 minutes prior to HPLC analysis.

54 CHAPTER V

SUMMARY AND CONCLUSION

In the assessment of overall acceptance of sweetness, color and mouthfeel on 18 juice mixtures, the overall analysis of sweetness was significant, F (17,170) = 6.99, P<.0001. The rating scale used was 1-9; 9-like extremely and 1-dislike extremely. The 1:3 grape-apple combination had the most overall acceptance, while 3:1 pomegranate-cranberry mixture had the least overall sweetness acceptability. The overall analysis of color was significant, F (17,170)

=3.05, P<.0001. The 1:1 grape-cranberry had the highest overall acceptance, while 1:3 pomegranate-apples had the least overall acceptability of color. The overall analysis of mouthfeel was significant, F (17,170) =10.08, P<.0001. The 3:1 grape-apple blend had the highest mean score of overall acceptance of mouthfeel 7.54 ± 0.85 while 3:1 pomegranate- cranberry blend had the lowest mean score of overall acceptance of mouthfeel 4.41 ± 1.99.

There was no significant difference among the six 1:1 juice combinations for color preference at P< 0.05. The 1:1 grape-cranberry mixture ranked the highest in color preference with a mean score 4.36 ± 0.97. However, 1:1 pomegranate-apple combination ranked the lowest in color preference, 2.77 ± 1.26. There was an overall significance at P<0.05 among the six 1:3 juice mixtures for color preference. The 1:3 grape-cranberry mixture had the highest mean score in color preference, 4.82 ± 1.10, while 1:3 pomegranate-apple blend had the lowest ranking in color preference, 2.41 ± 0.71. There was no overall significant difference in the ranking of color among the six 3:1 juice combinations at P<0.05. The 3:1 grape-cranberry combination had the highest mean score in color preference, 4.32 ± 0.95, while 3:1 pomegranate-apple blend had the lowest mean score in color preference, 2.86 ± 1.53.

55 There was an overall significance in sweetness preference of the six different 1:1 combinations of juice at P<0.05. The 1:1 grape-apple mixture had the highest mean score in sweetness preference, 4.77 ± 1.12. The 1:1 pomegranate-cranberry blend had the lowest score in sweetness preference, 2.09 ± 0.81. There was an overall significance in sweetness preference of the six different 1:3 blends of juice at P<0.05. The 1:3 grape-apple mixture ranked the highest in sweetness preference with a mean score of 4.36 ± 1.12. The 1:3 grape-pomegranate mixture ranked the lowest in sweetness preference with a mean score of 2.32 ± 1.60. There was an overall significance in sweetness preference ranking of the six different 3:1 combinations of juice at P<0.05. The 3:1 grape-apple mixture ranked the highest in sweetness preference with a mean score of 4.91 ± 0.80. The 3:1 pomegranate-apple mixture had the lowest score in sweetness preference, 1.5 ± 0.56.

There was an overall significance in mouthfeel preference ranking of the six different 1:1 combinations of juice at P<0.05. The ranking scale used was 1-6; 6-most preferred and 1-least preferred. The 1:1 grape-apple combination ranked the highest in mouthfeel preference with a mean score of 5.23 ± 1.02. The 1:1 pomegranate-cranberry blend had the lowest score in mouthfeel preference, 1.68 ± 0.64. There was an overall significance in mouthfeel preference ranking of the six different 1:3 combinations of juice at P<0.05. The 1:3 grape-apple mixture ranked the highest in mouthfeel preference with a mean score of 4.55 ± 1.69, while 1:3 grape- pomegranate mixture had the lowest score (1.90 ±1.44) in mouthfeel preference. There was an overall significance in mouthfeel preference ranking of the six different 3:1 mixtures of juice at

P<0.05. The 3:1 grape-cranberry blend had the highest mean score in mouthfeel preference, 5.23

± 0.29, and therefore, ranked the highest in mouthfeel preference. Nevertheless, 3:1 pomegranate-apple mixture ranked the lowest with a mean score of 1.77 ± 0.70.

56 The results of the sensory evaluation by the panelists showed that the purple grape- cranberry blend was the most preferred based on color in the three concentrations, 1:1, 1:3, and

3:1 while pomegranate-apple combination was the least preferred. The purple grape-apple blend was most preferred for sweetness and mouthfeel for all concentrations although for 1:1 sweetness and 3:1 mouthfeel the purple grape-cranberry combination was most preferred. In addition, purple grape-apple mixture had the highest rating in overall acceptance based on sweetness and mouthfeel, while purple grape-cranberry blend rated highest in overall acceptance of color. These results do not support the third hypothesis which stated that “a combination of pomegranate and cranberry juice will be the most acceptable juice combination as identified by the sensory panel”.

Purple grape juice was in all the blends that ranked high in all preferences and also rated highest in the overall acceptance. On the other hand, pomegranate juice was present in all blends that ranked the lowest in preference and also in the overall acceptance. This outcome does not support the fifth hypothesis which stated that “The blend of juices that will be most acceptable to the panelists will also have the highest concentration of ellagic acid”. Pomegranate juice had the highest concentration of ellagic acid, 103 mg/L, while the other individual juices ranged from 1 mg/L to 2 mg/L, which supports the second hypothesis which stated that “Pomegranate juice will have the highest concentration of ellagic acid when compared to the other individual juices in this study”. However, the result failed to support the first hypothesis which stated that “A combination of pomegranate and cranberry juice will have a higher concentration of ellagic acid than each juice alone”. Nevertheless, the 3:1 pomegranate-cranberry combination had the highest ellagic acid concentration, 97 mg/L, compared to the other juice mixtures as shown in Table 5. In

57 addition, hydrolyzed pomegranate juice had a higher ellagic acid concentration as compared to the unhydrolyzed juice.

In a study conducted by Gil and others (2000), a single strength form of pomegranate juice is said to contain 1561 mg/L of punicalagins, 121 mg/L ellagic acid, and 417 mg/L of other hydrolysable tannins [Table 6]. In another study conducted by Seeram and others (2004), 180 mL of pomegranate juice (in concentrate form) was reported to contain 139 mg/L of free ellagic acid and 1767 mg/L of ellagitannins. These studies show different ellagic acid concentrations depending on how the juice was processed. There is a significant change in the ratio of concentrations suggesting a variation based on different methods of processing. This may contribute to this study’s result not matching published results. The ellagic acid concentration in the juices with lower concentrations was enhanced by combining those juices with pomegranate juice. Nevertheless, the juices with pomegranate as a component in the blends ranked the lowest in both preference and overall acceptance. Purple grape juice had the highest ºBrix of 17.3, while pomegranate had 16.9 ºBrix, which failed to support the fourth hypothesis which stated

“pomegranate juice will have the highest ºBrix as compared to other juices in this study”.

Table 6-Phenolic compound composition (mg/L) of pomegranate juices Pomegranate juicesa Phenolic compounds 1 2 3 4 Gallagyl-type tannins punicalagin B 12.7 14.4 421.3 434.9 punicalagin D 10.1 11.1 838.5 918.2 other 45.1 102.5 302.0 525.6 Total gallagyl-type tannins 67.9 128.1 1561.7 1878.8 Ellagic acid derivatives ellagic acid glucoside 17.9 17.9 83.2 91.3 ellagic acid 15.3 8.7 37.9 172.8 Total ellagic derivatives 33.2 26.5 121.1 264.0 a(1)Juice from fresh arils; (2) juice from frozen arils; (3) single-strength commercial juice; (4) commercial juice from concentrate. Commercial juices are extracted by crushing whole pomegranates. (Source: Gil and others 2000)

In conclusion results from this study suggested that the juices that ranked or rated high in color, sweetness and mouthfeel did not have high concentrations of ellagic acid. To improve the sweet-bitter taste of pomegranate juice, which may have been the cause for all pomegranates’ combination lower ranking and overall acceptance, another study should be conducted using pomegranate juice that has undergone more clarification or filtration in order to improve the taste. Alternatively, the present study can be repeated using two types of pomegranate juice, one processed with the husk and the other processed without the husk. Also, a study where distilled water is a component of the juice mixtures used in this study should also be conducted, and the results compared to those of this study to determine whether water improves the overall acceptability of pomegranate juice. A study may be conducted to investigate the ºBrix effect on the acceptability of the juice combination used in the present study. Future study involving a larger sample of panelists should be designed to compare the results. In addition, the juices used in this study should be further analyzed to determine if they contain phytochemicals that interact synergistically with ellagic acid in the prevention of chronic diseases.

59 REFERENCES

Ahmed S, Wang N, Hafeez BB,Cheruvu VK, Haqqi TM. 2005. Punica granatum L. extract

inhibits IL-1β-induced expression of matrix metaloproteinases by inhibiting the activation of

MAP kinases and NF-kB in human chondrocytes in vitro. J Nutrition 135:2096-2102.

Anon. 2004. Average brix values and dominant acids. Warrensburg. MO. Available from:

. Accessed 15 November 2006.

Barth SW, Fahndrich C, Bub A, Dietrich H, Watzl B, Will F, Briviba K, Rechkemmer G. 2005.

cloudy apple juice decreases DNA damage, hyperproliferation and aberrant crypt foci

development in the distal colon of DMH-initiated rats. Carcinogenesis 26(8):1414-21.

Block G, Patterson B, Subhar A. 1992. Fruit, vegetables and cancer prevention: A review of the

epidemiological evidence. Nutr. Cancer 8(1):1-29.

Boyer J, Liu RH. 2004. Apple’s phytochemicals and their health benefits. Nutr Journal

3(5):1475-2891.

Cerd´a B, Llorach R, Ceron JJ, Espin JC, Tomas-Barberan FA. 2003. Evaluation of the

bioavailability and metabolism in the rat of punicalagin, an antioxidant polyphenol from

pomegranate juice. Eur J Nutr 42:18-28.

Dew TP, Day AJ, Morgan MRA. 2005. Xanthine oxidase activity in vitro: Effects of food

extracts and components. J Agric Food Chem 53:6510-15.

Falsaperla M, Morgia G, Tartorone A, Ardito R, Romano G. 2005. Support ellagic acid therapy

in patients with hormone refractory prostate cancer on standard chemotherapy using

vinorelbine and estramustine phosphate. Eur Urology 47:449-55.

60 Ferguson PJ, Kurowska E, Freeman DJ, Chambers AF, Koropatnick DJ. 2004. A flavonoid

fraction from cranberry extract inhibits proliferation of human tumor cell lines. J Nutr

134:1529-1535.

Gil MI, Tomas-Barbaran FA, Hess-pierce B, Holcroft DM, Kader AA. 2000. Antioxidant

activity of pomegranate juice and its relationship with phenolic composition and processing. J

Agric Food Chem 48(10):4581-9.

Ignarro LJ, Byrns RE, Sumi D, de Nigris F, Napoli C. 2006. Pomegranate juice protects nitric

oxide against oxidative destruction and enhances the biological actions of nitric oxide. Nitric

Oxide 15(2):93-102.

Keevil JG, Osman HE, Reed JD, Folts JD. 2000. Grape juice, but not orange juice or grapefruit

juice, inhibits human platelet aggregation. J Nutr 130:53-56.

Knekt P, Kumpulainen J, Järvinen R, Rissanen H, Heliövaara M, Reunanen A, Hakulinen T,

Aromaa A. 2002. Flavonoid intake and risk of chronic diseases. Am J Clin Nutr 76(3):560-

568.

Lansky EP, Harrison G, Froom P, Jiang WG. 2005. Pomegranate pure chemicals show possible

synergistic inhibition of human PC-3 prostate cancer cell invasion across matrigel. Invest New

Drugs 23:121-122.

Lee JH, Johnson JV, Talcott ST. 2005. Identification of ellagic acid conjugates and other

polyphenolics in muscadine grapes by HPLC-ESI-MS. J Agric and Food Chem 53:6003-10.

Li TM, Chen GW, Su CC, Lin JG, Yeh CC, Cheng KC, Chung JG. 2005. Ellagic acid induced

p53/p21 expression, G1 arrest and apoptosis in human bladder cancer T24 cells. Anticancer

Res 25(2A):971-9.

61 Liu RH. 2003. Health benefits of fruit and vegetables are from additive and synergistic

combinations of phytochemicals. Am J Clin Nutr 78(3):517S-20S.

Liu RH, Liu J, Chen B. 2005. Apples prevent mammary tumors in rats. J Agric Food Chem 53

(6):2341-43.

Losso JN, Bansode RR, Trappey A, Bawadi HA, Truax R. 2004. In vitro anti-proliferative

activities of ellagic acid. J Nutr Biochem 15:672-678.

Meilgaard M, Civille GV, Carr BT. 1987a. Sensory evaluation techniques. Vol. I Florida. CRC

press. 47- 112 p.

Meilgaard M, Civille GV, Carr BT. 1987b. Sensory evaluation techniques. Vol. II Florida. CRC

press. 29- 110 p.

Miguel G, Dandlen S, Antunes D, Neves A, Martins D. 2004. The effect of two methods of

pomegranate juice extraction on quality during storage. J Biomed and Biotech 5:332-37.

Moskowitz H. 1988. Applied sensory analysis of foods. Vol I Florida.CRC press.39 p.

Narayanan BA, Geoffroy O, Wilmingtom MC, Re GG, Nixon DW. 1999. P53/p21 (WAFI/CIP

1) expression and its possible role in G1 arrest and apoptosis in ellagic acid treated cancer

cells. Cancer Lett 136 (2):215-21.

Narayanan BA, Narayanan NK, Stoner GD, Bullock BP. 2002. Interactive gene expression

pattern in prostate cancer cells exposed to phenolic antioxidants. Life Sci 70:1821-39.

National Research Council. 1982. Committee on Diet, nutrition, and cancer. National Academy

Press, Washington, D.C.

National Research Council. 1989. Diet and health: implications for reducing chronic disease risk.

National Academy Press, Washington, D.C.

62 Oregon State University. 1998. Available from:

. Accessed 6 June 2007.

Pantuck AJ, Leppert JT, Zomorodian N, Aronson W, Hong J, Barnard RJ, Seeram N, Liker H,

Wang H, Elashoff R, Heber D, Aviram M, Ignarro L, Belldegrun A. 2006. Phase II study of

pomegranate juice for men with rising prostate-specific antigen following surgery or radiation

for prostate cancer. Clin Cancer Res 12 (13):4018-26.

Scalbert A, Williamson G. 2000. Dietary intake and bioavailability of polyphenols. J Nutr

130:2073S-85S. Available from: < http://jn.nutrition.org/cgi/content/full/130/8/2073S/F1>.

Accessed 23 October 2006.

Seeram N, Lee R, Hardy M, Heber D. 2005. Rapid large scale purification of ellagitannins from

pomegranate husk, a by- product of the commercial juice industry. Separation and purification

technology 41:49-55.

Seeram N, Lee R, Hardy M, Heber D. 2004. Bioavailability of ellagic acid in human plasma after

consumption of ellagitannins from pomegranate (punica granatum L.) juice. Clinica Chimica

Acta 348:63-68.

Seeram NP, Adams LS, Zhang Y, Lee R, Sand D, Scheuller HS, Heber D. 2006b. Blackberry,

black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts inhibit growth

and stimulate apoptosis of human cancer cells in vitro. J Agric. Food Chem 54:9329-39.

Seeram NP, Henning SM, Zhang Y, Suchard M, Zhaoping L. 2006a. Pomegranate juice

ellagitannin metabolites are present in human plasma and some persist in urine for up to 48

hours. J Nutr 136:2481-2485.

Shaheen SO, Sterne JAC, Thompson RI, Songhurst CE, Margetts BM, Burney PGJ. 2001.

Dietary antioxidants and asthma in adults. Am J Respir Crit Care Med 164(10):1823-1828.

63 Siegel GJ, Agranoff BW, Albers RW, Fisher SK, Uhler MD. 1999. Basic neurochemistry

molecular, cellular and medical aspects. 6th ed. Lippincott Williams and wilkins. Available

from: < http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=bnchm.section.3441>. Accessed 6

June 2007.

Soong YY, Barlow PJ. 2006. Quantification of gallic acid and ellagic acid from longan seed and

mango kernel and their effects on antioxidant activity Food Chem 97:524-30.

Stein JM, Keevil JG, Wiebe DA, Aeschlimann S, Folts JD. 1999. Purple grape juice improves

endothelial function and reduces the susceptibility of LDL cholesterol to oxidation in patients

with coronary artery disease. Circulation 100:1050-55.

Stone H, Sidel JL. 2004. Sensory evaluation practices. San Diego. Elsevier.53 p.

Sumner MD, Elliott-Eller M, Weidner G, Daubenmier JJ, Chew MH, Marlin R, Raisin CR, Dean

O. 2005. Effects of pomegranate juice consumption on myocardial perfusion in patients with

coronary heart disease. Am J Cardiol 96:810-14.

Sun Y. 1990. Free radicals, antioxidant enzymes, and carcinogenesis. Free Radic Biol

Med 8(6):583-99.

Rangkadilok N, Worasuttayangkurn L, Bennett RN, Satayavivad J. 2005. Identification and

quantification of polyphenolic compounds in longan (Euphoria longana Lam) fruit. J Agric

Food Chem 53:1387-92.

Robinson JW, Frame EMS, Frame II GM. 2005. Undergraduate instrumental analysis.6th ed.

New York: Marcel dekker. 806 p.

Thompson FE, Midthune D, Subar AF, McNeel T, Berrigan D, Kipnis V. 2005. Dietary intake

estimates in the National Health Interview Survey, 2000: Methodology, Results, and

Interpretation. J Am Diet Assoc 105:352-63.

64 Urquiaga I, Leighton F. 2000. Plant polyphenol antioxidants and oxidative stress. Biol Res 33(2):

55-64.

Vattem DA, Ghaedian R, Shetty K. 2005. Enhancing health benefits of berries through phenolic

antioxidant enrichment: focus on cranberry. Asia Pac J Clin Nutr 14(2):120-130.

Vattem DA, Jang H-D, Levin R, Shetty K. 2004. Synergism of cranberry phenolics with ellagic

acid and rosmarinic acid for antimutagenic and DNA-protection functions. J Food Biochem

30(1):98-116.

Wada L, Ou B. 2002. Antioxidant activity and phenolic content of Oregon cranberries. J. Agric.

Food Chem 50:3495-3500.

Weaver MC, Daniel RJ. 2003. The Food Chemistry Laboratory. 2nd ed. Florida: CRC press.

6 p.

Wilson T, Porcari JP, Harbin D. 1998. Cranberry extract inhibits low density lipoprotein

Oxidation. Life Sci 62(24):381-86.

APPENDIX A: CONFIDENTIAL QUESTIONNAIRE

66 Pre-Screening Questionnaire for Juice Sensory Panel

Please Print

Name:

Date:

Telephone No:

Gender: Female Male

Age:

Time: 1. On the time schedule attached, please indicate which time you will be available to participate on a regular bases.

Health: 1. Do you smoke? Yes No

2. If yes, how many packets of cigarettes do you smoke per day?

3. Do you take any medications that may affect your senses, especially taste and smell?

Yes No

4. Are you diabetic? Yes No

5. Do you have a cold or flu that may affect your taste senses? Yes No

6. Are you allergic to any fruit juice (s)? Yes No

If yes, please list the juice (s)

7. To your knowledge, have you had an injury that has affected your taste or smell? Yes No

8. Do you drink alcohol? Yes No

(Modified from Meilgaard et al 1987, Vol II pg 54)

APPENDIX B: CONSENT FORM

Consent to Serve as a Participant in a Sensory Panel

I have been asked to serve as a volunteer participant in a taste panel which is being conducted by Caroline Kamau, a graduate student from the School of Family and Consumer Sciences. The project will take two months; in which the participants will be required to participate approximately 5 hours a week based on the individual’s availability.

The purpose of this taste panel is to determine the overall acceptability of commercially prepared and combinations of these juices. The juices will be evaluated based on sweetness, color and mouthfeel. You will complete a screening test to determine your ability to discriminate differences among several taste stimuli presented. You will be presented with four coded but unidentified products for familiarization purposes. You will then be presented with a randomly numbered set of eight samples, of which a subset will be identical to the initial set tasted. You will be asked to identify the samples that are similar to the initial set. You will then participate in two triangle taste tests.

This screening will help determine your ability to detect differences among similar products with ingredient variables. In this phase of participant screening, each individual will be presented four different groupings of three solutions. Two of the solutions in each group will be distilled water and the other a sucrose solution. The concentration of the sucrose solution will be increased with each grouping. You will be asked to select the different sample in each grouping and to continue through each successive grouping until the taste of the different sample could be identified as one of the four basic taste modalities that are sweet, salty, sour, or bitter. The above procedure will be repeated using citric acid solution with a sour taste as the different solution. The results of the above test will be analyzed and if you score > 75%, you will qualify to participate in the studies panel.

If you qualify as a study taste panelist, you will participate in two preference ranking tests and one hedonic rating scale (see the ranking and rating scale test forms attached).

The study data are confidential. Your informed consent form will be placed in an envelope and kept in a locked drawer in 413 Johnson hall. Once the questionnaires are used to determine the potential taste panel participants, they will then be put in an envelope and kept together with the consent forms in a locked drawer. You will be assigned an ID code which will be used on all evaluation forms to maintain your confidentiality.

The nature of the project has been clearly explained to me by Caroline Kamau. I have been informed that the anticipated risks are no greater than those normally encountered in drinking commercially prepared fruit juices in daily life. However, as in all research, there may be unforeseen risks. If an accidental injury occurs, appropriate emergency measures will be taken. The participant will gain limited sensory evaluations skills by the end of the project, and be given a $ 10 gift certificate upon completion of all phases of the sensory evaluation part of the study.

I agree to participate on the understanding that I may terminate my participation at any time i so desire. I have been informed that participation in this study is voluntary and that my decision to participate or not to participate will not impact grades/ class standing/ relationship to the institution. If you have a question or any problems regarding this project; please contact Caroline Kamau at (419)-372-7825 email: [email protected] or Dr. Julian Williford at 419-372-7833 email: [email protected]. If you have any questions or concerns about participant rights, you may contact the Chair, HSRB Joseph Jacoby at 419-372-7716 email: [email protected].

Signature of participant Date

APPENDIX C: SENSORY SCREENING TEST

Matching Test I Form

Name: Date:

Instructions: Please taste the first set of pink coded samples; rinse your mouth with the provided water. Taste the second set of green coded samples and determine which samples in the second set correspond to a sample in the first set. Write down the code of the sample in the second set next to its match from the first set.

First set Second set match

981

194

229

371

Discrimination Test Form

Name: Date:

Instructions: Please taste the samples presented to you from left to right. Two samples are identical one is different. Select the different sample and indicate by placing an X next to the code of the different sample.

Sample code Indicate the different sample

737

932

895

(Modified from Meilgaard et al 1987, Vol I pg 51)

72 Finally, the individuals were required to taste four solutions, representing each of the basic tastes

(sucrose- sweet, sodium chloride- salty, citric acid- sour, quinine sulfate- bitter) they had to identify each correctly to qualify for the taste panel (Meilgaard and others, 1987, Vol II pg 49).

Matching Test II Form

Name: Date:

Instructions: Please taste the coded samples from right to left; rinse your mouth with the provided water. Match the four tastes (sweet, salty, sour and bitter) with the right code. Write down the taste of the sample next to its match from the first set.

First set Second set match

748

651

426

374

(Modified from Meilgaard et al 1987, Vol II pg 47)

73 Table 7- Potential panelists screening test results Participants screening results Participants code Score 0F2001 83% 0M1002 100% 0M1003 *67% 0F1004 100% 0F2005 92% 0M1006 83% 0M1007 75% 0F1008 92% 0M1009 75% 0F10010 100% 0F10011 92% 0M10012 83% 0M20013 75% 0M10014 83% 0M10015 75% 0F20016 83% 0F10017 92% *This potential panelist was disqualified since he did not make the 75% pass mark.

APPENDIX D: SENSORY EVALUATION SCORE CARD

75 Hedonic Rating Scale Test Form

Product

Code: Date

Instructions: The panel will be asked to rate the juices as follows:

Please check the appropriate response which reflects your overall acceptance of sweetness for each sample.

Table 8- Hedonic rating scale test

Ratings 148 897 539

Like Extremely

Like Very Much

Like Moderately

Like Slightly

Neither Like or Dislike

Dislike Slightly

Dislike Moderately

Dislike Very Much

Dislike Extremely

(Modified from Weaver and Daniel 2003, p.39)

76 Ranking Test Form

Product

Code: Date

Instructions: Please rank the six samples (683, 429, 662, 353, 768, and 418) in descending order for color preference.

Most preferred

Least preferred

(Modified from Meilgaard et al 1987, pg 105)

Ranking Test Form

Product

Code: Date

Instructions: Please rank the six samples (148, 897, 539, 575, 272, and 244) in descending order for color preference.

Most preferred

Least preferred

(Modified from Meilgaard et al 1987, pg 105)

APPENDIX E: HPLC ANALYSIS

78 Table 9- Replicate results of HPLC analyses of individual juices and juice blends Sample Area 1 Area 2 Ave area Standard deviation Hydrolyzed pom (P) 167.9880 158.5880 163.8900 6.646804 Unhydrolyzed pom (UP) 44.2840 55.9070 50.1000 8.218702 Cranberry (C ) 3.4460 2.6330 3.0400 0.574878 Apple (A) 2.7510 3.3000 3.0300 0.388202 Purple Grape (G) 1.9280 1.5710 1.7500 0.252437 1/2 :1/2 Combination GC 1.0760 1.4440 1.2600 0.260215 GA 0.7590 0.8310 0.8000 0.050912 GP 92.8300 88.2260 90.2600 3.255520 PC 80.1500 86.2730 83.2100 4.329615 CA 2.2220 3.3260 2.7700 0.780646 PA 88.7150 90.5530 89.6300 1.299662 1/4:3/4 Combination GC 1.3920 2.3910 1.8920 0.706400 GA 3.2500 3.4100 3.3300 0.113137 GP 123.9940 122.3500 123.1720 1.162484 PC 57.3560 59.1170 58.2365 1.245215 CA 1.7260 2.0840 1.9050 0.253144 PA 54.3260 54.1730 54.2495 0.108180 3/4:1/4 Combination GC 0.9200 1.0680 0.9940 0.104652 GA 1.5190 1.4550 1.4868 0.045538 GP 46.4910 46.5780 46.5345 0.061518 PC 161.9370 145.8840 153.9105 11.351190 CA 1.8660 2.0844 1.9752 0.154432 PA 153.6190 149.9770 151.7980 2.575283

Ellagic acid has a low solubility in water, typically about 25 mg/liter, and the solubility in

40-50% methanol water mixtures is comparable. The standards were prepared at concentrations

of 150-300 mg/liter in methanol, and the working standards were prepared by quantitative

dilution using a solvent that produced standards in 40-50% methanol/water. These studies

included standards and samples (pomegranate hydrolysis products) that ranged in concentration

up to 10-75 mg/liter. The calibration graph, using freshly prepared standards, was linear. When

standards at 30-75 mg/l were observed after 5-24 hours, there were white sediments (precipitated

ellagic acid). If a precipitate formed during the first few hours, the particles were too small to be

79 detected even using a laser beam. Presumably, this sample remained homogeneous when sampled, and the fresh mobile phase quickly dissolved any crystals that reached the column. This may have resulted in a slight broadening (or tailing) of the HPLC peak, but it did not invalidate the calibration curve. Independent tests with diluted pomegranate samples also showed that no ellagic acid was lost in the preparation and sampling of these samples.

In pomegranate juice, the largest fraction of ellagic acid is bound with tannins. The ellagotannins were hydrolyzed to ellagic acid by heating in 1 M hydrochloric acid for 30 minutes. Tests showed that longer heating did not produce additional hydrolysis product. The total ellagic acid in the hydrolyzed sample was reported. Physiologically the ellagotannins are hydrolyzed to ellagic acid, so the total is the appropriate quantity.

The preliminary work tested a solid phase extraction (SPE) technique for cleaning up samples. This was intended to remove some additional components, especially those that might exhibit long retention time and prolong analytical runs. The SPE method also provides an opportunity to concentrate samples.

a. The C18-Low 1000 mg/6 mL columns (GracePure SPE) were used.

b. The column was conditioned with methanol, and then the aqueous sample was

drawn through the column.

c. The column was rinsed with water, and the combined aqueous layers were

discarded.

d. The ellagic acid and other component was extracted by two 5 ml additions of

methanol. These were combined, diluted to volume, and analyzed as the sample.

The solid phase extraction (SPE) technique was abandoned since the aqueous extract retained measurable amounts of ellagic acid.