Pueraria Lobata) And

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Determination of physicochemical and sensory properties of kudzu (Pueraria lobata) and

potato starch in beef patties, and thermal stability of kudzu root extract

isoflavones in beef patties

Shweta Kumari

A Dissertation Submitted to the Faculty of Mississippi State University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Food Science in the Department of Food Science, Nutrition and Health Promotion

Mississippi State, Mississippi

December 2012

Shweta Kumari

2012

Determination of physicochemical and sensory properties of kudzu (Pueraria lobata) and

potato starch in beef patties, and thermal stability of kudzu root extract

isoflavones in beef patties

Shweta Kumari

Approved:

______James M. Martin Juan L. Silva Assistant Professor of Food Science, Professor and Graduate Coordinator, Nutrition and Health Promotion of Food Science, Nutrition and Health (Co-Major Professor) Promotion (Co-Major Professor)

______Janice DuBien J. Byron Williams Associate Professor of Mathematics and Assistant Professor of Food Science, Statistics Nutrition and Health Promotion (Minor Professor) (Committee Member)

______Sandra Lynn B. Burney R. Hartford Bailey Instructor of Food Science, Nutrition Professor of Pathobiology/ and Health Promotion Population Medicine (Committee Member) (Committee Member)

______George M. Hopper Dean of the College of Agriculture and Life Sciences

Name: Shweta Kumari

Date of Degree: December 15, 2012

Institution: Mississippi State University

Major Field: Food Science

Co-Major Professors: Dr. Mike Martin and Dr. Juan L. Silva

Title of Study: Determination of physicochemical and sensory properties of kudzu (Pueraria lobata) and potato starch in beef patties, and thermal stability of kudzu root extract isoflavones in beef patties

Pages in Study: 133

Candidate for Degree of Doctor of Philosophy

Kudzu (Pueraria lobata) plant is an edible leguminous vine. This study focused

on the utilization of kudzu starch and kudzu root extract in beef patties. We hypothesized that a) physicochemical and sensory properties of beef patties formulated with kudzu starch, is comparable to those of potato starch; b) the kudzu root extract is rich in isoflavones, and isoflavones quantity is not affected during cooking. In Study I, beef patties were formulated using modified commercially available kudzu and potato starch each at 2.0, 4.0 and 6.0% (wt/wt). Starch treated beef patties were compared with respect to change in physical, chemical, color, textural and consumer responses as affected by starch type (kudzu, potato) and starch level (2, 4, 6 %). Additionally starch treated patties were compared to all-beef patties. Kudzu starch treated patties were significantly lower in moisture % (62.7 vs. 64.4), higher in fat % (9.1 vs. 8.3), protein % (26.3 vs. 24.7), hardness (9.3, vs. 6.9 N) and gumminess (3.7 vs. 1.9 N) compared to potato starch treated patties. Starch treated samples were significantly lighter in color and had lower (P <0.05) expressible moisture compared to all-beef patties. Patties with 6% kudzu or potato starch

were significantly higher in cooking yield than all-beef patties. No significant difference

existed in consumer overall liking scores of kudzu or potato starch treatments and control

beef patties with no added starch. The overall liking scores ranged between 5 ‘neither

like nor dislike’ and 6 ‘like slightly’ for all samples. In study II, kudzu root extract was

prepared, and using HPLC, ten isoflavones were detected with puerarin and daidzein

accounting for 95% of the total isoflavones. Beef patties were formulated with kudzu root

extract at 0, 1, and 3% (wt/wt), and four isoflavones were detected in uncooked and

cooked patties, considering other isoflavones diluted to undetectable levels in patties.

Results indicated that cooking did not change the amount of isoflavones in beef patties.

This study illustrates the characteristics of kudzu starch compared to conventionally used potato starch in meat model system and verifies the thermal stability of isoflavones in beef patties.

Key words: Kudzu root starch, kudzu root extract, beef patties, isoflavones

DEDICATION

I dedicate this manuscript to my parents, Mrs. Indira Singh and Mr. Jaikant Singh.

They have been by my side when the times have been tough. Mom and Dad, I would have never reached this far without you, I love you both dearly and can never thank you enough! I also dedicate this work to my sisters and brothers, Sushmita and Swati,

Shivendra and Suhanshanu for being there at every step of my life. I cannot think of

myself without you guys.

ACKNOWLEDGEMENTS

I would like to sincerely acknowledge my gratitude to several people without whom, this work would have never been completed. First of all, I would like to thank my major professor, Dr. Mike Martin, whose technical guidance, and a very straightforward practical approach led me to become a leader, a moderator and a team worker. At this point, when I look back, I remember the summer of 2006, when I had known Dr Martin only for 10 days, and I had to undergo a major surgery, Dr Martin was very calm, supportive and an involved person then, and he has been the same caring person ever since. Words can never justify my gratitude and respect towards him as a mentor. I also owe heartfelt gratitude to the Mississippi Beef Council for funding the project.

I would like to express my deepest gratitude to Dr. Patti C. Coggins, who had a major contribution of ideas in formulating the basics of this study. Dr. Coggins has a charismatic personality and her company always filled me with dreams, hope, confidence and positive energy. I owe my thanks to Dr. Douglas Marshall, his expert, experienced and intellectual queries and advice helped me to fine tune my study.

Thanks are also due to my committee members, Dr. J. Byron Williams, Dr. Lynn

Burney, and Dr. R. Hartford Bailey, and Dr. Juan Silva for their expert guidance with this research. I remember the long discussion hour with Dr. Bailey on my qualifier answers, and at the end, led me to think “people who did great service to society were not

‘different’ but common people who dared to listen to their hearts.

iii

Dr. Janice Dubien, as far as I know, I have taken all the courses that she offers,

yet I would love to be in her class over and over again. Her course layout, teaching style,

perfect examples of each experimental design, thorough step by step approach to

problems, correction and marking style at the same time, strict and lovable personality

make her one of my ideal teachers of my life. As a minor professor she cleared all my doubts and guided me with research analysis. I look forward to applying her guidance in the future with analysis and interpretation.

I would like to thank Dr. Jose M. Rodriguez and Mr. Joey Raines, from

Mississippi State Chemical Laboratory who provided infrastructure and guided the HPLC part of my study. I approached Dr. Rodriguez on one busy fall day with my proposal for kudzu root extract analyses, and given the intensive time involved and limited funding from my grant, I wasn’t very hopeful. However, he just asked me one question, “you want to do this….?” I said “yes!”, “ok, then we can do this” and from then onwards it became an easy path for me. Joey taught me the basics of HPLC at the same time pushing my study forward.

I would like to express my thanks to Mr. Tim Armstrong, and Mr. James Cannon, who made possible the implementation of this study in the meat lab. I thank Mr. William

Monroe, Institute for Imaging and Analytical Technologies, Mississippi State University who assisted in the Scanning Electron Microscopy part of this study.

I would also like to thank Ms. Julie Wilson, Vijay Radhakrishnan, Monil Desai and others at Food Science Nutrition and Health Promotion Department for all of their assistance with various parts of my research. I also thank Mr. Joseph Andol for being a great friend and guide. Overall, I thank everyone mentioned here and others who have

made permanent imprints on my life and made this research and my journey of 6 years in

Starkville a memorable one.

Last, but not least, I would like to express my gratitude to my husband and the love of my life, Shyamesh Kumar, for being by my side through all the ups and downs and challenging moments of my life.

TABLE OF CONTENTS

DEDICATION ...... ii

ACKNOWLEDGEMENTS ...... iii

LIST OF TABLES ...... ix

LIST OF FIGURES ...... xi

CHAPTER

I. INTRODUCTION ...... 1

II. LITERATURE REVIEW ...... 3

2.1 Kudzu ...... 3 2.1.1 Background ...... 3 2.1.2 Plant description...... 4 2.1.2.1 Roots ...... 4 2.1.2.2 Stem ...... 5 2.1.2.3 Leaves ...... 5 2.1.2.4 Flowers ...... 6 2.1.2.5 Fruit ...... 6 2.1.3 Nutritional value ...... 6 2.1.4 Medicinal uses ...... 7 2.1.5 Culinary uses ...... 8 2.2 Kudzu root starch ...... 9 2.2.1 Chemistry and Structure ...... 10 2.2.2 Gelatinization ...... 11 2.2.3 Retrogradation...... 13 2.2.4 Rheology ...... 15 2.3 Meat and Starch ...... 16 2.3.1 United States Regulations for beef products ...... 18 2.4 Kudzu root extract ...... 19 2.5 Sensory evaluation ...... 22 2.5.1 Consumer Analysis /Affective test ...... 23 2.5.2 Quantitative Descriptive Analysis ...... 24

III. EFFECTS OF KUDZU ROOT STARCH AND POTATO STARCH ON PHYSICAL, CHEMICAL AND SENSORY PROPERTIES OF BEEF PATTIES ...... 26

3.1 Introduction ...... 26 3.2 Materials and Methods ...... 28 3.2.1 Meat processing and patty preparation ...... 28 3.2.1.1 Cooking of patties ...... 30 3.2.2 Physical analysis ...... 30 3.2.2.1 Cooking yield ...... 30 3.2.2.2 pH ...... 31 3.2.2.3 Expressible moisture ...... 31 3.2.3 Chemical analysis ...... 31 3.2.4 Color evaluation ...... 32 3.2.5 Texture profile analysis ...... 33 3.2.6 Scanning electron microscopy (SEM) ...... 33 3.2.7 Sensory evaluation ...... 34 3.2.7.1 Sensory Sample Preparation ...... 34 3.2.7.2 Consumer Acceptability...... 35 3.2.8 Statistical analysis ...... 35 3.3 Results and Discussion ...... 38 3.3.1 Physical analysis ...... 39 3.3.1.1 pH ...... 39 3.3.1.2 Cooking yield ...... 40 3.3.1.3 Expressible moisture ...... 40 3.3.2 Chemical analysis ...... 42 3.3.2.1 Moisture content ...... 42 3.3.2.2 Fat content ...... 43 3.3.2.3 Protein content ...... 44 3.3.3 Color evaluation ...... 49 3.3.4 Texture profile analysis ...... 51 3.3.5 Scanning electron microscopy ...... 55 3.3.6 Sensory evaluation / Consumer acceptability ...... 64 3.4 Conclusions ...... 71

IV. QUALITATIVE AND QUANTITATIVE ESTIMATION OF ISOFLAVONES IN KUDZU ROOT EXTRACT AND BEEF PATTIES CONTAINING VARIOUS LEVELS OF KUDZU ROOT EXTRACT USING HPLC ...... 73

4.1 Introduction ...... 73 4.2 Materials and Methods ...... 76 4.2.1 Reagents ...... 76 4.2.2 Preparation of stock and calibration solutions ...... 77 4.2.3 Plant sample preparation ...... 77 4.2.4 HPLC analysis ...... 78 vii

4.2.5 Validation of HPLC method, identification of peaks and calculations of isoflavones content ...... 79 4.2.6 Calculations: ...... 80 4.2.7 Basic hydrolysis of kudzu root extract...... 81 4.2.8 Acid hydrolysis of kudzu root extract ...... 81 4.2.9 Preparation and cooking of beef patties with kudzu root extract ...... 81 4.2.9.1 Cooking of patties ...... 82 4.2.10 Extraction of isoflavones from uncooked and cooked beef patties ...... 83 4.3 Statistical analysis ...... 84 4.4 Results and discussion ...... 84 4.4.1 Identification and quantification of isoflavones in kudzu root extract ...... 84 4.4.2 Stability of Kudzu root extract isoflavones in beef patties during cooking ...... 92 4.5 Conclusions ...... 96

V. CONCLUSION ...... 97

REFERENCES ...... 101

APPENDIX

A. SAS OUTPUTS ...... 109

A.1 SAS outputs for Table 3.2, Table 3.4, Table 3.7, Table 3.9 and Table 3.11 ...... 110 A.2 SAS outputs for Table 3.3, Table 3.8, Table 3.10 and Table 3.12...... 122

B. APPROVED INSTITUTIONAL REVIEW BOARD (IRB) FORM ...... 127

C. INFORMED CONSENT FORM ...... 129

D. SCORE SHEET FOR CONSUMER ACCEPTABILITY TEST ...... 131

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LIST OF TABLES

2.1 Nutritional value of kudzu leaf, stem and root (Corley and others 1997) ...... 7

3.1 Treatment codes and formulations...... 28

3.2 Analysis of variance on the effects of starch type and starch level on physical properties of beef patties formulated with kudzu and potato starch ...... 39

3.3 pH (uncooked), cooking yield and expressible moisture (cooked) of beef patties formulated with and without kudzu or potato starch...... 41

3.4 Analysis of variance on the effects of starch type and starch level on chemical properties of beef patties formulated with kudzu and potato starch ...... 45

3.5 Fitted Regression Equations as a function of level of starch on physicochemical properties of beef patties formulated with kudzu or potato starch...... 48

3.6 Proximate composition of uncooked and cooked beef patties formulated with and without kudzu or potato starch ...... 49

3.7 Analysis of variance on the effects of starch type and starch level on color parameters of uncooked beef patties formulated with kudzu and potato starch ...... 50

3.8 CIE L*, a*, b* color mean values of uncooked beef patties formulated with and without kudzu or potato starch...... 51

3.9 Analysis of variance on the effects of starch type and starch level on textural properties of beef patties formulated with kudzu and potato starch ...... 52

3.10 Texture profile analysis of cooked beef patties formulated with and without kudzu or potato starch...... 53

3.11 Analysis of variance on the effects of starch type and starch level on consumer acceptability of beef patties formulated with kudzu and potato starch ...... 66

3.12 Consumer acceptability of cooked beef patties formulated with and without kudzu or potato starch 1, 2, 3 ...... 67

3.13 Agglomerative Hierarchical Clusters (AHC) of beef patties with and without kudzu or potato starch based on consumer overall liking 1, 2 ...... 68

4.1 Isoflavone content (μg/ml dry weight) in (a) kudzu root crude extract; (b) kudzu root extract after acid hydrolysis; (c) kudzu root extract after basic hydrolysis ...... 91

4.2 Effect of cooking on isoflavone content (μg/gm, dry weight) in uncooked and cooked beef patties formulated with kudzu root crude extract at 0%, 1% and 3%...... 93

LIST OF FIGURES

2.1 Stem, leaf, flower (SEPPC2009), and root tuber of kudzu (Simoons 2002) ...... 5

2.2 Chemical Structure of (a) amylose and (b) amylopectin (Zamora 2007)...... 11

3.1 Fitted curve of moisture content as a function of starch level for kudzu and potato starch cooked patties...... 46

3.2 Fitted curve of fat content as a function of starch level for kudzu and potato starch cooked patties...... 46

3.3 Fitted curve of protein content as a function of starch level for kudzu and potato starch cooked patties...... 47

3.4 Scanning electron microscopy images of uncooked beef patties with no starch and no water (Negative Control) ...... 57

3.5 Scanning electron microscopy images of potato starch ...... 59

3.6 Scanning electron microscopy images of kudzu starch ...... 60

3.7 Scanning electron microscopy images of uncooked beef patties with 6% potato starch and 5% water (P6) ...... 61

3.8 Scanning electron microscopy images of uncooked beef patties with 6% kudzu starch and 5% water (K6) ...... 63

3.9 Agglomerative Hierarchical Clusters- C1, C2, C3, C4 illustrating group of consumers (n=159) with different preference patterns based on overall liking scores of beef patties with and without kudzu or potato starch...... 68

3.10 Internal preference map of consumers’ liking scores of the beef patties with and without kudzu or potato starch ...... 70

4.1 HPLC chromatogram of A) standard solution; B) kudzu root crude extract ...... 86

4.2 Scheme of acid and basic hydrolysis of isoflavone derivatives (Delmonte and Rader 2006) ...... 88

4.3 HPLC chromatogram of A) kudzu root crude extract after acid hydrolysis; B) kudzu root crude extract after basic hydrolysis ...... 89

4.4 HPLC chromatograms of uncooked beef patties...... 94

4.5 HPLC chromatograms of cooked beef patties...... 95

xii

CHAPTER I

INTRODUCTION

Kudzu (Pueraria lobata) is an edible leguminous vine of the family Fabaceae.

For more than 2,000 years, Japanese and Chinese cultures have found great value in kudzu (Keung 2002). All parts of the kudzu vine contain valuable components. The roots serve as a source of fine natural food and contain specialty starch, and strong adsorbent fibers which are useful in making paper. Kudzu vines are suitable for making textiles.

Kudzu root is the most widely utilized part, it is used to produce high quality cooking starch and is also known to contain several isoflavones, some of which may be used as phytoestrogens (Delmonte and Rader 2006; Choi and Ji 2005). Because of the high amounts of these compounds present in kudzu, it is used in several commercial dietary supplements advertised as nutraceuticals.

A prized plant of the far East (Japan and China), kudzu is widely acknowledged as a noxious plant in the United States. Kudzu was introduced in the United States in

1876 at the Centennial Exposition in Philadelphia and promoted as an ornamental and livestock feed. Later in the 1930s - 40s kudzu was promoted as a method to control soil erosion, and in the six years from 1935 to 1940, the US Soil Conservation Service made available 73 million seedlings to landholders for erosion control (USDA 2010; USDA and ARS 2009; Shurtleff and Aoyagi 1977). Kudzu is presently growing on over six million acres in the southern parts of United States causing significant economic loss in 1

terms of decrease in forest products and the cost of removal of kudzu vines. An alternative cost effective strategy to control kudzu would be to utilize the plant to develop consumer acceptable food products for commercial production and leveraging its international demand to market products.

This research focused on utilization of kudzu root starch and kudzu root extract as a functional and nutraceutical ingredient in a meat product. Meat, a staple in the

American diet was chosen as a delivery system for kudzu starch and extract. The refined starch obtained from the root has been incorporated into numerous traditional and folk recipes of soups, sauces, creams, clear soups, jelled salads, jellied confections, jelled desserts, thickened beverages, noodles and as a coating for deep fried vegetables and in the making of kudzu noodles (Shurtleff and Aoyagi 1977). However, there are no documented studies or previously performed research that reports its functionality or consumer testing in beef patties. Starch has historically been added to processed meat products to increase yield, bind moisture, provide heat and shear stability, extend shelf life, and improve freeze/thaw stability and texture (Taagart 1996). Although kudzu root is reported to contain 10-fold more isoflavone than soybeans, (Delmonte and Rader 2006;

Choi and Ji 2005), no studies have used kudzu root extract in food products.

The objectives of this research were to: 1) compare the physical, chemical, textural and sensory properties of kudzu starch at levels of 2, 4, and 6% to potato starch at levels of 2, 4, and 6% in beef patties; and 2) identify and quantify isoflavones from kudzu root and to assess the isoflavones thermal stability in beef patties. .

CHAPTER II

LITERATURE REVIEW

2.1 Kudzu

2.1.1 Background Kudzu is a vine that belongs to the plant kingdom in the pea family Fabaceae in the genus Pueraria. According to the classification, genus Pueraria has two species,

Pueraria montana and Pueraria phaseoloides. The most common species, Pueraria montana, has two varieties Pueraria montana var. lobata (Willd.) and Pueraria montana var. montana (Taiwan kudzu or tropical kudzu). Kudzu, Pueraria montana var. lobata

(Willd.), has synonyms such as Dolichos lobatus, Pueraria hirsuta, Pueraria lobata,

Pueraria lobata thomsonii and Pueraria thunbergiana (USDA 2010). Kudzu is a coarse,

high climbing, twining, perennial vine considered native to Japan and China where it is

used as a medicinal plant and as forage for animals. The name ‘kudzu’ derives from the

Japanese word kuzu or 葛 or クズ.

In 1876, the United States celebrated the first World’s fair, and the 100th

anniversary of the signing of the Declaration of Independence in Philadelphia,

Pennsylvania. Kudzu was brought to this International Centennial celebration by

Japanese representatives to exhibit flowering plants and to promote an ornamental plant

useful for livestock feed. During the 1930s and 1940s, kudzu was recommended for erosion control, and in the six years from 1935 to 1940, the US Soil Conservation Service 3

made available 73 million kudzu seedlings to landholders for erosion control. The

southern climate, with its hot humid summers, frequent rainfalls and absence of long

freezing winters became more conducive for the growth of kudzu than Japan and China.

The plant grew profusely covering 150,000 acres of land annually and tolerated

herbicides used for control. In 1953, the United States government officially declared

kudzu a noxious weed, thus shifting the interest of study to eradication. The highest

infestations are in Alabama, Georgia, and Mississippi (Everest and others 1999; USDA

and ARS 2009). Kudzu has been nicknamed the ‘foot-a-night vine’, ‘mile-a-minute’, and

‘the vine that ate the South’ (USDA and ARS 2009).

2.1.2 Plant description

Kudzu (Pueraria montana lobata) is a semi-woody vine which reaches up to 30 m (98 ft) in length. The plant can be described in terms of roots, stem, leaves, flowers and fruit (Figure 2.1).

2.1.2.1 Roots The kudzu root stores carbohydrates and typically grows to 1-3 m (3-9 ft) in length below ground and up to 17.8 cm ( 7 in) in diameter (Southeast Exotic Pest Plant

Council (SEPPC) 2009). In Japan, the kudzu root is mostly used in two forms, white

refined kudzu root powder (starch) used in several beverages and creamy soups, and unprocessed root pieces simmered with other herbs of medicinal function to alleviate

intestinal or digestive disorders (Shurtleff and Aoyagi 1977). In addition to the

carbohydrate (starch) application, the cellulose fibers available in the roots are utilized in

paper-making. Several other chemicals are extracted and used for drug applications.

Figure 2.1 Stem, leaf, flower (SEPPC2009), and root tuber of kudzu (Simoons 2002)

2.1.2.2 Stem The vines in the early stages have diameters approx. 1.3 cm (0.5 in) and can reach up to 10 cm (4 in) in older patches (SEPPC2009). The strong fibers of the vines are used in textiles.

2.1.2.3 Leaves Leaves are alternate (leaves attachments are singular on stem in different directions with 180º angle in between) and compound (trifoliate) meaning the blade/ lamina of leaf is divided into three leaflets (Figure 2.1). Leaflets are up to 10 cm (4 in) across in size. The leaflet and stem has hairy margins which are more pointed and piercing in young leaves than older ones. The leaves are similar to spinach, collards and

other table greens, and can be eaten or consumed as a tea (Keung 2002). However, commercial use of kudzu leaves as a salad green is not yet common in the United States.

2.1.2.4 Flowers The kudzu plant starts to flower in the third year and blooms between July and

October. The flowers are purple, fragrant and about 1.3 cm (0.5 in) long, similar to pea flowers. Some traditional uses of the flowers are as an ingredient in jams and jellies

(Simoons 2002).

2.1.2.5 Fruit Seeds are found in flat, 5 cm (2 in) long, hairy pods. One pod contains three hard seeds which mature between September and October (Simoons 2002).

Thus, all parts of P. lobata contain valuable items to be used. Irrespective of the fact that each part is edible, there are at least 30 valuable chemicals for potential drugs identified in P. lobata (Shurtleff and Aoyagi 1977).

2.1.3 Nutritional value

Corley and others (1997) evaluated the nutritional analyses of kudzu (Pueraria lobata) in root (tuber parts), leaf and stem and as a feed for ruminants. Table 2.1 shows the amount of crude protein, NDF (neutral detergent fiber), ADF (acid detergent fiber),

Ca, Fe, K and Mg contained in the kudzu plant parts. Li and others (1998) analyzed and studied the water, starch, five major minerals and seventeen amino acid content in fresh roots of kudzu and in commercially available kudzu powder. The starch content in root powder was 39.1% compared to 2.4-28.3% in the fresh root. Other nutrients like Ca, Fe,

P, Zn and Mg were more abundant in kudzu than in other foods like sorghum and wheat;

however, the contents were more prevalent in fresh wild kudzu roots than in the kudzu powder.

Table 2.1 Nutritional value of kudzu leaf, stem and root (Corley and others 1997)

Parameter Leaf Stem Root

Crude protein (% of dry matter) 17.5 10.3 8.6

Neutral-detergent Fiber (% of dry matter) 48.1 73.7 39.8

Acid-detergent Fiber (% of dry matter) 38.2 44.0 53.3

Ash (% of dry matter) 8.3 7.9 4.3

Ca (% of dry matter) 0.7 0.1 0.4

Fe (mg kg-1) 162.3 156.6 3,600

K (% of dry matter) 1.0 1.0 0.3

Mg (% of dry matter) 0.3 <0.1 0.1

2.1.4 Medicinal uses Various studies have been conducted to investigate purported medicinal qualities of folk remedies using kudzu. Kudzu root extract is reported to control hypertension, migraine headaches, and sudden deafness by the improvement of cerebral circulation

(Qicheng 1980). The plant is also applied as an antipyretic, antidiarrhetic, spasmolytic, diaphoretic, antiemetic and antidipsotropic agent (Keung 2002; Lai and Tang 1989).

Pharmacological studies have shown kudzu root extract suppressed voluntary alcohol intake and alcohol withdrawal symptoms in rats receiving free access to water and alcohol. The kudzu root extract contained the isoflavones puerarin, diadzin, diadzein,

genistin, genistein and glycetin (Benlhabib and others 2004). Isoflavones have structural

similarity to β- estradiol and thus its health benefits have been evaluated in age-related and hormone-dependent diseases (Hendrich and Murphy 2001a; Delmonte and Rader

2006). Jun and others (2003) compared the antioxidative activities of isoflavones from kudzu root (Pueraria labata ohwi). Diets rich in antioxidants are considered to reduce the risk of chronic disorders such as DNA damage, mutagenesis, carcinogenesis, and atherosclerosis by protecting the biological system against free radicals (Jun and others

2003). There has also been a study on evolution of the isoprene biosynthetic pathway in kudzu (Sharkey and others 2005). The biological pathway by which isoprene is emitted by certain species of plants into the atmosphere helps maintain atmospheric chemistry.

The emitted isoprene is converted by free radicals available in the atmosphere into epoxides, which combine with water to form aerosols and haze. This isoprene pathway protects plants from environmental stress conditions like temperature variation, ozone and other reactive oxygen species (Sharkey and others 2005). Today, kudzu extracts are available to consumers as dietary supplements mostly containing isoflavones from kudzu.

Additional kudzu potential is yet to be discovered and utilized in the areas of research, medicine, and culinary arts.

2.1.5 Culinary uses In the United States, corn starch, potato starch and wheat starch are the most widely used cooking starches because of the low price and the availability compared to kudzu starch. Unlike kudzu starch, corn and wheat starch are not a whole foods, since the life-giving germ of the corn and wheat kernels is removed before wet milling; this process increases shelf life, but causes loss of vital nutrients and flavor (Shurtleff and 8

Aoyagi 1977). Kudzu starch can be used in sauces, creams, clear soups, jelled salads, jellied confections, jelled desserts, thickened beverages, as a coating for deep fried

vegetables and in the making of kudzu noodles. Kudzu leaves and stems could be

steamed, boiled to form a tea, sautéed, deep fried or pickled. Kudzu flowers can be used

to make jam, jellies or pickled with vinegar. Kudzu once was considered a staple along

with brown rice, millet, broomcorn and buckwheat in Japan. Kudzu powder has been

recommended by Japanese naturopaths as a replacement for grains as the primary staple

due to its low caloric value and reasonably low price (Shurtleff and Aoyagi 1977).

2.2 Kudzu root starch Starch is a major component of root and tuber crops grown throughout the world.

Root and tuber crops contain 70-80% water, 16-24% starch and trace quantities (<4%) of

proteins and lipids. Some of the edible roots and tubers are: potato, sweet potato, cassava,

arrowroot, lotus, kudzu and colocassia (Hoover 2001). Starch has its own unique quality

that exists in granular form. Starch granule size, shape, chemical composition, and

molecular organization vary not only among different botanical sources, but also among

genotypes. Native starches are obtained by extracting the undamaged purified form of

starch from other components of the raw material. Native starches are used as thickeners

and stabilizers in food products. Modified starch is processed using heat, alkali, acid, bleach, high shear, low pH or modified using other techniques or chemicals to improve starch functionality in food manufacturing processes. The Food and Drug Administration regulates the use of the various modified food starches by stipulating the types of modification allowed, degree of modification and the reagents used in chemical modification. The food label is required only to state that "modified starch" is present. 9

There are resistant starches which escapes digestion in the small intestine of healthy

individuals (Sajilata and others 2006).

Kudzu starch is categorized as a starch from a non-conventional source due to its

non-popularity in food products and commercial use (Takeiti and others 2007). However,

if the unique properties of kudzu starch are investigated and practical applications in food

processing are initiated, the kudzu curse of the southern United States could be turned

into a boom or blessing. The unique properties of kudzu (P. lobata) starch make it a

superior starch. The yield of kudzu starch ranges from 65-162 g starch/kg depending on

the age of the root (Achremowicz and Tanner 1996). Kudzu starch is more cohesive in

gel form than other starches. It is more elastic, resists crumbling and presents gel stability

superior to other natural starches due to its temperature-time-viscosity characteristics.

Kudzu gels do not liquefy but release water upon boiling. The kudzu gels do not permit

the separation or crystallization of liquids during freeze-thaw cycles. Kudzu starch gels

give a faint sweet taste to the tongue with no aftertaste and dissolves rapidly (Keung

2002).

2.2.1 Chemistry and Structure Starch is made up of two major components amylose and amylopectin. Amylose is a minor component that consists mainly of α-(1→4) linked D- glucopyranosyl residues

with a molecular weight ranging from 105 to 106 daltons (Da). Amylopectin is the major

component with molecular weight of the order 107 – 109 Da. It is composed of linear

chains of (1- >4)-α- D- glucose residues connected through (1→6)-α-linkages (5-6%)

(Figure 2) (Hoover 2001).

Figure 2.2 Chemical Structure of (a) amylose and (b) amylopectin (Zamora 2007).

Kudzu starch is reported to have 20.8 % - 21% of amylose and 20.5 chain length

(CL) of amylopectin. Kudzu starch was found to contain 124 ppm of phosphorous mostly in amylopectin (Suzuki and others 1981). The fat and protein content is about 0.15 and

0.08% respectively. The physicochemical properties are different from potato, corn and wheat starch (Achremowicz and Tanner 1996). Achremowicz and others also found that

80% of the kudzu starch granules had an average diameter of less than 32 microns. The scanning electron microscope (SEM) images revealed that kudzu granules are short, sharp edged and angular compared to corn and lotus starch (Geng and others 2007).

2.2.2 Gelatinization Gelatinization is an important characteristic of starch. Starch has the ability to change physical properties of many foods which makes it very valuable to the food

industry. When starch granules are dispersed in excess water and heated, the intermolecular bonds of the starch granules break exposing the hydrogen bonding sites to water. This process is called gelatinization. Starch undergoes a phase change from an ordered to a disordered state with the uptake of heat, the loss of crystallinity and increased viscosity (Li and others 2004). Some researchers (Ghiasi and others 1982;

Miller and others 1973) found that the increase in viscosity does not entirely depend on the solubility of starch. They concluded that as more water was bound, the concentration of soluble starch in the remaining free water increased, causing increased viscosities.

Thus, starch can be used as a thickener, for gelling and as a binding agent due to its gelatinization properties.

Temperature is an important determinant of function during gelatinization. When native starches are heated in the presence of water and the temperature rises to a critical value, the birefringences of the native starch granules are lost. this critical temperature known as gelatinization temperature (Li and others 2004). Onset temperature is referred to as the temperature at which the first granule loses birefringence and final temperature is when more than 95% of the granules loose birefringences. Gelatinization can be measured by microscopic analysis, viscosity based measurements and thermal analysis.

Microscopic analysis includes polarized light microscopy with a heating stage. Viscosity based measurements are based on the principle of change in viscosity under controlled mixing, heating and cooling. The instruments employed for this measurement include visoamylograph and rapid visco analyser (RVA). Under thermal analysis, differential scanning calorimetry (DSC) is used for measurement of gelatinization temperature and enthalpy. This is a popular method among researchers, as the measurement process can

be accurately adjusted to change moisture content, irrespective of viscosity

measurements, where the suspension concentration has to be lower than 10% (percentage

differs with the species of starch). However, there are drawbacks to this method such as

the method expense and the difficulty of working with suspensions at lower temperatures

due to the fast sedimentation of starch granules in cold water. Some researchers (Li and others 2004; Wang and Sastry 2000) have employed ohmic heating (change in electrical

conductivity) to determine the starch gelatinization temperature.

Geng and others (2007) compared gelatinization temperatures among lotus starch, kudzu starch and corn starch using DSC. They found gelatinization onset temperature

(To) of kudzu starch (60°C) similar to corn starch (60.67°C). The gelatinization

temperature range Tc (conclusion temperature) - To) of kudzu starch was 21.97°C, lotus

starch 13.65°C and corn starch 9.35°C. DSC parameters are considered to be influenced

by the molecular architecture of the crystalline region, which corresponds to the amylose

- amylopectin ratio (Noda and others 1996). Srichuwong and others (2005b) studied 15

different native starches using DSC and found that the onset gelatinization temperature of

kudzu starch was 68.4°C, corn 62.6°C, rice 61.6°C and potato 61.4°C. The temperature

range (onset temperature – conclusion temperature) of kudzu, corn, rice and potato was

19.8°C, 18.7°C,18.4°C and 16.3°C respectively.

2.2.3 Retrogradation When starch is added to water to form a paste or heated with water to form a gel,

the amylose and amylopectin crystalline structure is lost. When the gel or paste

undergoes freezing or thawing, the linear parts of amylose and amylopectin rearrange to

form a more crystalline structure. This process is called retrogradation. Retrogradation 13

forces water out of polymer network resulting in syneresis. Starch retrogradation is the

major factor in the staling of bread and other baked products. Retrogradation depends on

various factors like temperature, concentration, type of starch and presence of other

ingredients (Takeiti and others 2007).

Retrogradation can be measured using different techniques such as x-ray

diffraction analysis, thermal methods like differential scanning calorimetry (DSC) and

rheological techniques. X-ray diffraction analysis looks at the change in the pattern of

amylose and amylopectin. The thermal process works on the principle that the transition

enthalpy (heat change during each phase change) increases progressively in magnitude

with storage time until a certain limit is reached and remains constant thereafter. Aged

gels or stale bread show a characteristic melting endotherm (absorbing heat) around 55-

60°C that is absent in fresh gels and breads immediately after gelatinization (Eliasson

1996). Differential scanning calorimetry is suitable to measure retrogradation of starch pastes but it is not suitable to measure gels because syneresis is difficult to measure.

Rheological techniques, mostly viscoelastic measurements are well suited to determine gel firmness (rigidity) on aging. There are other methods also employed to examine retrogradation such as enzymatic digestion, quantitative centrifugation, Raman

spectrometry and the current NMR (nuclear magnetic resonance) technique (Karim and

others 2007).

Kudzu starch contains a C-type x-ray diffraction pattern. The C type starch is

highly susceptible to environmental temperature, similar to the starches of soybean and

sweet potato (Suzuki and others 1992). In general, cereal starches such as corn have an

A-type diffraction patterns like corn starch (Geng and others 2007). The X-diffraction

pattern also showed that kudzu contained both spherical (Hung and Morita 2007) and polygonal granules (Srichuwong and others 2005b).

2.2.4 Rheology The rheological properties of starches can be studied using a Brabender viscoamylograph and rotational viscometers (Hoover 2001) and the rapid visco-analyzer

(Srichuwong and others 2005a). During the gelatinization process and the conversion of

starch paste to gel, the crystalline structures of starch are disrupted by increased hydrogen

bonding between water molecules and hydroxyl groups, and this bonding induces

granular swelling. The loss of free water and the restricted flow of water due to

enormously swollen granules occupying more space, contribute to the increased viscosity

of the starch gel. These swelling properties of starch provide important characteristics to

food products and their rheological behaviors. Srichuwong and others (2005) studied the

swelling and pasting properties of 15 native starches from different plant origins, and

showed that kudzu starch has different properties than other starches. Kudzu starch had

an amylopectin unit- chain (APC) ratio of 0.393, corn 0.392, potato 0.364 and rice 0.449.

They reported that the swelling power of starch granules increased with the APC ratio.

Achremowicz and Tanner (1996) compared the viscosity of kudzu starch with potato and corn starch using Brabender viscoamylograph in Brabender Units (BU) and found that kudzu starch viscosity was two-fold lower than the viscosity of potato and corn starches, and somewhat closer to the viscosity of wheat starch. The findings in this study about the properties of kudzu starch could offer a novel use in starch applications.

The consistency, flow rate and viscosity of a semi-solid product can be measured by a Bostwick consistometer (BC) and a Brookfield viscometer. The Bostwick 15

consistometer is a simple, inexpensive instrument used to monitor product consistency in a wide range of foodstuffs. However, limitations include operator variability and subjectivity, leveling and dryness of the instrument, and serum separation at the edge of product flow. Laboratory viscometers are also widely used as quality control instruments based on simple rotational viscometry, with the shear rate dependent upon the spindle type and the rotation speed used. There are several viscometers available for process control, including tube, rotational and vibrational viscometers which are suitable to the specific texture properties of products in the food industry (Cullen and others 2000).

2.3 Meat and Starch Starch is second to cellulose in natural abundance in plants and is the stored energy reserve (Taagart 1996). Today starch is recognized as one of the most versatile ingredients used in the food industry. To use starch as an ingredients with optimum functionality, the properties of starch need to be understood based on end-product attributes. Acid, sugar, and fats are the three co-ingredients that need to be considered before selecting a starch for a product formulation. In a low pH food, the acid disrupts naturally occurring hydrogen bonding causing the starch granule to swell and premature rupture of granules with decreased viscosity. Thermally treated starch works under low pH conditions. Sugar raises the gelatinization temperature of starch which makes it more difficult to cook-out the starch and achieve the desired functionality. Sugars can be added after starch is cooked or pre-gelatinized starch can be used. In the presence of fats or oils, the viscosity of starch does not increase due to the coating of starch granules and delaying of hydration. Lipophilic starches are used as emulsion stabilizers and to ensure product quality and stability (Taagart 1996). Starch granules are prone to rupture and 16

breakdown under high temperature, longer hold time and greater shear forces. In such

cases, modified granules for thermal treatment are best suited. Starch is used in a wide

variety of ready-to-eat preprocessed and frozen products. Starch must maintain its

characteristics through freezing and thawing with low retrogradation in order to preserve

the final quality of the product. Starch is used in baked goods, batters and breadings,

beverage emulsions, flavor encapsulation, confectionery, dairy products, fruit

preparations, gravies, soups and sauces, mayonnaise and salad dressings, savory snacks

and meat products (Taagart 1996).

In preprocessed meats, starch acts a as water binder to increase yield, reduce

cooking losses, improve texture, sliceability and succulence, and increase shelf life. Most

reformed or ground meats are required to be cooked to a minimum internal temperature

of 72-75°C for product safety. For a product with no acid or shear, starches with low

gelatinization temperature (such as potato) are recommended. Waxy-maize and tapioca

starches have a high degree of stabilization, low gelatinization temperature and an

excellent water binding temperature necessary for frozen or chilled products. Pre-

gelatinized starch in reformed or comminuted meat binds water quickly, increasing the

viscosity, and enhancing a more open, coarse structure (Taagart 1996). In meat, the

protein network has the role of binding water. During gelatinization, the starch uses water

to form a gel and thus retains the juiciness and palatability of the product. There are

various researchers (Berry and Wergin 1993; Shih and Daigle 2003; Warner and others

2001) who have added starch in meat products as fat replacers to improve water-binding,

add bulk, and improve texture and mouthfeel. Hsiao and others (1999) reported that fat reduction can be achieved with the use of starch in products like taco meat, spaghetti and

meat balls while maintaining consumer acceptability. Sodium alginate, in combination

with modified tapioca starch evaluated in low-fat beef patties, showed an improvement in

juiciness, tenderness and cooking yields without increasing the fat retention and without

effecting the beef flavor (Berry 1997). Nisar and others (2009) also used tapioca starch

with low-fat buffalo patties and found a significant improvement in processing, composition and sensory properties. When four fat replacers, carrageen gum, modified cassava starch, microparticulated whey protein and oat bran were compared to control beef frankfurters with pork back fat, it was observed that cassava starch was similar in sensory acceptability to the control while each starch significantly reduced the total lipids

(Sampaio and others 2004). Aktas and Genccelep (2006) compared gelatinized, solubilized/dispersed and retrograded form of corn and potato starch with sheep tail fat in bologna-type sausage and reported that no difference in pH, water holding capacity, jelly

and fat separation among the modifications, except jelly and fat separation which differed

between potato and corn starches. Characteristics of low-fat (3% or less fat) beef patties

formulated with carbohydrate-lipid composites showed an increase in juiciness and

tenderness and a decrease in cohesiveness compared with 10% fat patties (Garzon and

others 2003). Kudzu starch with medium low gelatinization temperature appears to be a

potential non-conventional starch to be studied in beef patties.

2.3.1 United States Regulations for beef products There are certain terms which are used in this study are defined here in terms of

regulatory forms. According to the Code of Federal Regulations (CFR) Part 9, 319.15(c),

‘hamburger’ consists of chopped fresh and/or frozen beef with or without the addition of

beef fat and/or seasoning, and not more than 30 percent fat and no added water, 18

phosphates, binders, or extenders. ‘Beef patties’ can contain chopped fresh and/or frozen beef with or without the addition of beef fat and/or seasonings. Binders or extenders, spices, defatted beef patty tissue cab be used without added water or with added water only in amounts such that the product characteristics are essentially that of a meat pattie.

In terms of labeling a product, regulations state that a product can only be called ‘fat free’ if it contains less than 0.5gms of fat per labeled serving. A low fat product contains 3gms or less fat per 50gms of small serve size, and a reduced fat claim states that a product contains 25% percent less fat as compared to the appropriate reference food. A ‘percent fat-free’ or ‘percent lean’ claim is restricted to products that qualify as ‘low fat’. 90% fat free is synonym to 90% lean (USDA and FSIS 2007) .

2.4 Kudzu root extract Veterinarians have been reported estrogenic compounds in plants can induce estrus in immature animals or interfere with normal reproductive processes (Mazur and others 1998). More than 300 plants have been identified that possess contain compounds with estrogenic activity. These compounds are defined as phytoestrogens. Phytoestrogens comprise a variety of structurally diverse chemicals, with flavonoids as their largest group. Isoflavones (dietary phytoestrogens) are one class of flavonoids that are available in the seeds and other parts of various plant species mostly belonging to the Leguminosae family. Isoflavones have a similar structure to β-estradiol of mammals which allows it to reduce or activate estrogenic activity in the body by binding with the estrogen receptors on cells. Isoflavones have antioxidant properties comparable to vitamin E which can reduce the risk of cancer by preventing free radical damage to DNA. Isoflavones are protective against cancer, cardiovascular diseases, osteoporosis and various hormone 19

dependent conditions in women’s health (Choi and Ji 2005). Pueraria lobata has been

found to have an antiproliferative effect on human cancer cell lines. Pueraria radix is highly valued for medicinal properties (Fang and others 2005). Pueraria radix and its extracts are commercially available as dietary supplements (Prasain and others 2003).

The typical structures of isoflavone is a common 3-phenylchromen-2-one core structure and differs by substituents such as methoxy, hydroxyl and glycoside functions on the primary structure. Isoflavones are found in plants in the form of 6″-O-malonyl-7-

O-β-D-glucoside derivatives which is converted to 6″-O-acetyl-7-O-β-D-glucoside, 7-O-β-

D-glucosides or free isoflavones during food processing or sample preparation.

Commercial dietary isoflavone supplements generally contain soy, red clover or kudzu

extracts or a combination of these extracts. Each of these plants has a characteristic

isoflavone profile. Soybeans or soy foods contain primarily daidzein, glycitein and

genistein. Red clover has an abundance of formononetin and biochanin A. Kudzu root

has been shown to have 10-fold more isoflavone content compared to soybeans

(amounting to 2 g/ 100 g dry weight) (Choi and Ji 2005). Kudzu (Pueraria lobata

(Willd.) Ohwi mainly contains isoflavones such as puerarin (daidzein 7-C-β-D-glucoside),

daidzin (daidzein 7-C-β-D-glucoside), and daidzein (Rong and others 1998a). There are di-glucosides like daidzin-4′-O-glucoside and puerarin-4′-O-glucoside also extracted from kudzu root extract by LC-MS/MS (Liquid Chromatography- Mass spectrometry/

Mass Spectrometry) analysis (Delmonte and Rader 2006). In addition to phytoestrogenic

activity, kudzu root daidzin is also known to have inhibitory activity on cataldehyde

dehydrogenase, cAMP phosphodiesterase, and antispasmatic activity, whereas puerarin

has hypoglycemic effects and increased coronary blood flow (Choi and Ji 2005).

Several methods for the determination of isoflavonoids in kudzu have been reported, including, HPLC (Rong and others 1998a; Matkowski 2004; Kirokosyan and others 2003; Fang and others 2005), HPLC-tandem mass spectrometry (Zhang and others

2005) capillary electrophoresis (Chen and others 2001; Fang and others 2006b), HPLC and electrospray ionization tandem mass-spectrometry (Fang and others 2006a; Prasain and others 2003), HPLC –diode array detection –mass spectrometry (He 2000), and

HPLC-MS/ MS (Prasain and others 2007). Isoflavones can be extracted in alcohol by heating, refluxing method, chilled extraction method or just extracting at room temperature (Prasain and others 2007).

Kudzu root extract contains isoflavones and has antioxidant activity (Jun and others 2003). Several studies suggest that diets containing isoflavones with antioxidant activities are beneficial against cancer, aging, atherosclerosis and other free radical related diseases (Johnson and Loo 2000; Patel and others 2001; Mazur and others 1998).

The oxidation of lipids in food develops off flavors and chemicals harmful to health. The use of antioxidants in meat products can minimize oxidative deterioration. In the present market situation where consumers have an affinity and price-value to natural antioxidants, the food industry and researchers strive to meet the demand and focus on utilization of non-culinary/nutraceutical herbs or extracts potential to be a functional ingredient. Researchers have used ginger, cinnamon bark, licorice root, rehmania root, peony root and angelica root to mask flavor in goat meat (Kim and others 1993), and ethanol extracts of white peony, red peony, sappanwood, mountan peony, rosemary extract in ground beef to reduce lipid oxidation (Han and Rhee 2005). Rosemary extract is capable of reducing lipid oxidation and irradiation-induced quality changes in ready-to-

eat turkey bologna. Bloukas and others (1999) also observed increased overall acceptability of frankfurters formulated with extract of betanin (extract from beet juice) and paprika juice.

There are no documented studies or previously performed research available that reports use of kudzu root extract in beef patties. The sensory profile of kudzu root extract in food model system is not available. Studies on the sensory characteristics of soy isoflavones in food systems have documented the undesirable flavor characteristics of soy products (Uzzan and Labuza 2004). There is limited information available on kudzu root extract (Lau and others 2005; Rong and others 1998b) which contains isoflavone contents similar to soybean. Baik and others (2005) investigated the thermal stability of kudzu root isoflavones and changes in the amount of isoflavones in kudzu root extract by heating at 80, 100, 121, 140, 165 and 180°C for 90 min. They found that diadzin and genistin did not degrade or decrease in amount until 121°C was reached and it started to degrade thereafter. There seems to be various opportunities with kudzu root extract to be explored in food model systems.

2.5 Sensory evaluation Consumers expect a food that is safe, enjoyable to eat, nutritious and of consistent quality. If a food falls short of any of these criteria there is sufficient choice in a commercially competitive environment for consumers to change their allegiances and find alternative products. Food industry uses sensory science to minimize the risk of product failure (90% of new product launches fail), and to ensure that sensory quality is maintained in the long-term production (Kilcast 2011). Sensory science employs the senses of selected and trained human subjects to identify the individual perceptible 22

characteristics of food, and to quantify their intensities, while consumer science focuses on determining/predicting the overall acceptability of a product based on all the important senses like appearance, aroma, flavor and texture . Researchers have employed consumer

testing to determine the overall liking of meat formulations with various starch based fat

replacers such as- modified corn starch (Khalil 2000), corn starch (Garzon and others

2003), tapioca starch (Nissar and others 2009), sodium alginate (Berry 1997), bambara

groundnut (Vigna subterranean L.) (Alakali and others 2010), oat fiber (Pinero and others

2008), potato and corn starch (Aktas and Genccelep 2006), carrageen gum, modified

cassava starch, oat bran (Sampaio and others 2004), and combinations of polydextrose,

sugarbeet fiber, oat fiber, potato starch and pea fiber (Troutt and others 1992). There are

studies where starch based texturizers have been reported to improve texture, mouthfeel,

increase juiciness and tenderness of meat products (Aktas and Genccelep 2006; Berry

1997; Berry and Wergin 1993; Garzon and others 2003; Nissar and others 2009; Sampaio and others 2004; Shih and Daigle 2003; Warner and others 2001; Serdaroglu 2006; Troutt and others 1992). ASTM International, formerly known as the American Society for

Testing and Materials (ASTM) has standard methods for how to evaluate products through the human senses (sight, smell, taste, touch, and hearing).

2.5.1 Consumer Analysis /Affective test Consumer test measures preferences for products or magnitude of likes/dislikes for a product (Meilgaard and others 2007). Sensory consumer testing is an important technique in determination of the success of a new product in the market place. Two approaches can be used to evaluate consumer response. The first is a scientific experimental approach to measure consumer response to food products and get 23

statistically validated results. The second approach could be a qualitative approach through consumer interviews to provide insight and interpretation of human choices

(Meilgaard and others 2007).

Consumer tests can be an acceptance or/and preference test. Acceptance tests measure acceptability or liking of a food by a consumer. Preference tests measure the appeal of one food product over another. The most common method to quantify acceptance or measure preference of a product is the 9-point hedonic scale and the paired preference test, respectively. The 9-point hedonic scale has been used for years and is effective, however the 9-point, 3-, 5-, and 7-point facial scale are more useful for children

(Stone 1980). Consumer tests can be classified into two main categories on the basis of the primary task of the test:

Task Test and Type Questions

Choice Preference tests Which sample do you prefer?

Which sample do you like better?

Rating Acceptance tests How much do you like the product?

How acceptable is the product

2.5.2 Quantitative Descriptive Analysis Descriptive analysis is a sensory evaluation technique which involves detection or discrimination and description of both the qualitative and quantitative sensory aspects of the product by trained human subjects/panelists (Meilgaard and others 2007). The panelists should be able to detect and describe the perceived sensory attributes such as appearance, aroma, flavor and texture after the sample is smelled and tasted (Meilgaard and others, 2007). Commonly used descriptive test methods include the Flavor Profile 24

Method, the Texture Profile Method, the Quantitative Descriptive Analysis (QDA®)

Method and the Spectrum Descriptive Analysis Method. The QDA method was developed by Tragon Corp. and this method relies on statistical analysis to determine the appropriate terms, procedures and panelists that should be used for the analysis of a specific product (Stone and Sidel, 1992). Panelists are selected from a large pool of candidates according to their ability to discriminate differences in sensory properties of different samples. Panelists should be trained to gain experience in the specific descriptive method. The training of QDA panels requires the use of product and ingredient references for the development of a sensory language. The panel leader should not dominate or influence the group members. Attention is focused on consistent terminology development but panelists are free to develop their own approach while scoring on a 15 cm anchored (6 inch) line scale. QDA panelists evaluate the products individually in separate booths to reduce distraction and interaction among panelists. The results of QDA tests are statistically analyzed and the results are graphically represented in the form of a spider web with a branch or spoke from a central point for each attribute.

These techniques allow for development of sensory terminology or lexicon to define the sensory characteristics of food products.

CHAPTER III

EFFECTS OF KUDZU ROOT STARCH AND POTATO STARCH ON PHYSICAL,

CHEMICAL AND SENSORY PROPERTIES OF BEEF PATTIES

3.1 Introduction

Kudzu (Pueraria lobata) is an edible leguminous vine with medicinal properties.

Various parts of kudzu plants have been used in traditional herbal medicines and as a culinary ingredient. The powdered white starch prepared from kudzu root (a.k.a. Kudzu powder; Kuzu-ko in Japanese) is a fine quality cooking starch, which can be used as a gluten free thickening agent and as a coating for frying foods. Kudzu starch granules are fine in size, and are highly absorbent and stable among natural starches. The starch has lower gelatinization temperature and produces a colorless, odorless, soft transparent gel that is freeze/thaw stable, and it does not produce cloudiness or paper like starch residue.

It can be used to produce a gel in medicines and foods, including high protease containing foods such as pineapple and kiwi (Parks and others, 2002).

A prized plant of the far East (Japan and China), kudzu is considered a noxious plant in the United States. It is estimated to be growing on over six million acres of land in the southern United States, causing significant economic damage. Utilization of Kudzu in the form of consumer accepted food products (similar to Asian products) would have significant economic benefits. However, even though kudzu is safe and edible, it is not considered a mainstream food choice in the American diet. Most of the kudzu powder 26

sold in the United States is imported from Japan. The production and processing of kudzu starch in Japan takes about 60-90 days, which increases the final cost. In the United

States, an advanced processing facility could replace the repeated cleaning/ washing steps to reduce the cost of production and increase the yield. The average yield of starch is approximately 15-34% of the fresh roots (Hung and Morita 2007; Suzuki and others

1981). The need for research to explore the use of kudzu starch as a novel food ingredient for commercial market production is warranted.

Starches have been historically added to processed meat products to increase yield, bind moisture, provide heat and shear stability, extend shelf life, and improve freeze/thaw stability and texture of food products (Taagart 1996). The most desirable properties of starch used to improve meat performance include transparent texture, neutral-taste and comparatively lower gelatinization temperature. Potato starch is one of the earliest conventional starches used in meat systems. Desirable potato starch properties include high yield (22 to 52%), inexpensive production, bland flavor, lower gelatinization temperature (57-67°C), and high salt tolerance during cooking. These properties make it useful in meat products (Narpinder and others, 2005). The refined starch obtained from the kudzu root has been incorporated into numerous food products (soups, sauces, jelled desserts, noodles, as a coating for frying). However, there are no documented studies available that report its functionality or consumer testing in beef patties. The principle objective of this study was to compare the physical, chemical, textural, color and sensory properties of kudzu starch at 2, 4, and 6% to potato starch at 2, 4, and 6% in beef patties.

3.2 Materials and Methods The present study investigated the physical, chemical and sensory properties of

kudzu and potato starches in low fat beef patties. The experiment consisted of six

treatments (starch/water combinations) and two controls. Three replicates of the

experiment were run in three consecutive weeks. The treatments included, 3 levels (2, 4,

6%) of kudzu starch with 5% water, 3 levels (2, 4, 6%) of potato starch with 5% water, a

positive control with no starch and only 5% water, and a negative control with no starch

and no water. The list of the different treatments is outlined in Table 3.1.

Table 3.1 Treatment codes and formulations.

Treatment Treatment Description* Amounts of Starch + Water + Number Code Beef (gms) = Total 3405gms ~ 7.5 pounds 1 K2 2% K+ 5% W 68.10 + 170.25 + 3166.65 = 3405 2 K4 4% K+ 5% W 136.20 + 170.25 + 3098.55 = 3405 3 K6 6% K+ 5% W 204.30 + 170.25 + 3030.45 = 3405 4 P2 2% P+ 5% W 68.10 + 170.25 + 3166.65 = 3405 5 P4 4% P+ 5% W 136.20 + 170.25 + 3098.55 = 3405 6 P6 6% P+ 5% W 204.30 + 170.25 + 3030.45 = 3405 7 PC Positive Control (0% starch+ 0 + 170.25 + 3234.75= 3405 5% W) 8 NC Negative Control (0% 0 + 0 + 3405 = 3405 starch+ 0% W) *K, kudzu starch; P, potato starch; W, distilled water

3.2.1 Meat processing and patty preparation Fresh lean beef (Shoulder Clods, Institutional Meat Purchase Specifications

(IMPS) 114C) was obtained from the Meat Science Laboratory at the Department of

Food Science Nutrition and Health Promotion, Mississippi State University. A random

sample from each replication was analyzed to determine fat percentage using a near-

infrared spectrometer (FoodScan Lab Analyzer Model 78800, Foss Analytical, Eden 28

Prairie, MN) that is AOAC (Association of Official Analytical Chemists) approved

(Schilling and others 2001). The fat and moisture content ranged between 7-8 % and 68-

70%, respectively in fresh lean beef. Commercially available modified kudzu starch

(Mitoku Organic Wild Kuzu) was purchased from Natural Import Company, Biltmore

Village, NC; and modified potato starch (Eliane™ 100) was donated by Avebe Food Inc.,

Veendam, Netherlands. The fresh lean beef was ground using a 1.27 cm plate in a Hobart

meat grinder (80055 Mixer-grinder, Hollymatic Co., Countryside, IL). Eight 7.5 pound

batches of coarsely ground beef were randomly assigned to the six treatment and two

control groups. For each treatment (as described in Table 3.1), ground beef was hand

mixed for approximately 3minutes with different concentrations of kudzu or potato

starch, and water. Each treatment batch and controls consisted of precalculated amounts

of lean beef, kudzu starch, potato starch and water so that the total weight of each batch

was 3.60 kg (approximately 7.5 lbs). Kudzu starch or potato starch and water were hand

blended for approximately 2mins with the beef in different concentrations as indicated in

Table 3.1, followed by a second grinding through a 4 mm plate with a four blade knife in a Hobart meat grinder (80055 Mixer-grinder, Hollymatic Co., Countryside, IL) . The

different treatment batches were then stuffed (Risco I-36016 Thiene, Vincenza, Italy) into

7.62 cm diameter plastic tubes (Interstate Packaging, White Bluff, TN) and sealed at one end with metal clips to make logs of beef. The treatment beef logs were labeled and

frozen at -23°C until further evaluation. All equipment was cleaned between each

treatment for all replications. Exactly the same process was repeated to make samples for

the second and third replicates and the time between the replicates was 1 week.

3.2.1.1 Cooking of patties The frozen beef logs were removed from the plastic tubes and sliced into 1.27 cm

thick patties (Butcher Boy ™ American Meat Equipment, LLC, Selmer, TN) with

individual patties placed in a 3 mil plastic bag (Prime Source, 15.24 cm x 21.59 cm,

Kansas City, MO) and vacuum packaged (Model CV3HS, JVR Industries, Buffalo, NY)

at kPa (999 mbars). The frozen patties in vacuum packages were stored at 2.2°C

overnight. On the day of evaluation, sliced frozen patties were thawed at room temperature for 15 min, weighed and then cooked on a griddle top stove (Griddle 442A,

Toastmaster Inc., Booneville, MO). The griddles were preheated to 176°C for 5 min prior to placement of beef patties on the griddle. Each patty was cooked for 5 min on each side, with an additional 1 minute on each side until an internal temperature of 71°C was reached as measured by a handheld digital thermometer (Model PT100 RTD, Omega

Engineering Inc, Bridgeport, NJ). The uncooked patties were thawed at room temperature before measurements were feasible to be performed.

3.2.2 Physical analysis

3.2.2.1 Cooking yield The percentage cooking yield for each treatment for each replicate was determined by calculating the ratio of the weights of patties before and after cooking on three randomly selected patties from each treatment.

Percentage cooked yield = (cooked wt/ uncooked wt) x 100.

3.2.2.2 pH The pH of uncooked patties was obtained using portable pH meter (Accumet 61a,

Fisher Scientific, Hampton, NH). A 10 g sample of the uncooked patty was diluted to 100

ml with distilled water and then homogenized (Brinkmann homogenizer, Brinkman

Instruments Co., Westbury, NY) before evaluation. Three samples per treatment per

replicate were used for pH evaluation.

3.2.2.3 Expressible moisture The Instron Universal Testing Machine (Model 1011, Instron Corp., Canton, MA) was used to determine expressible moisture for four cooked patties from each treatment.

One 50 mm diameter core was removed from each patty. The cores were individually weighed and then placed between two, 12.5-cm diameters Whatman #1 filter paper sheets to absorb excess moisture. Cores were axially compressed to a height of 6.35 mm (to achieve 75% compression of original height of approximately 2.54 cm of individual cooked patty) and were held for 15 s until the deformation point was reached. After removing the load, the core was reweighed. The Instron was programmed with a 500-kg compression load cell and a crosshead speed of 100 mm/min. Expressible moisture was reported as a percentage:

Percentage expressible moisture = [(Initial wt – final wt)/initial wt)] × 100.

3.2.3 Chemical analysis Vacuum packaged frozen beef patties, obtained by slicing the frozen beef logs were held at 2.2°C overnight. Four uncooked and four cooked beef patties per treatment per replication were used to evaluate protein, fat and moisture percentage using a near-

infrared spectrometer (FoodScan Lab Analyzer Model 78800, Foss Analytical, Eden

Prairie, MN) that is AOAC (Association of Official Analytical Chemists) approved. The cooked patties were evaluated the same day of cooking. Four randomly selected uncooked patties were cooked as described above in the cooking of beef patties section

3.2.1.1 . The four randomly selected cooked patties were ground in a food processor

(Cuisinart, Mini-Prep Plus, 3-Cup Food Processor, New York, NY) separately and packaged tightly into two sample cups of 140-mm diameter, provided by FoodScan Lab

Analyzer to determine protein, fat and moisture content of the samples. The two determinations per treatment per replication for chemical analyses were used for statistical analysis.

3.2.4 Color evaluation The frozen patties obtained by slicing frozen logs made with each treatment were vacuum packaged (Model CV3HS, JVR Industries, Buffalo, NY) and left overnight at

2.2°C prior to evaluation, instrumental color determinations were made over the surface of vacuum packaged uncooked beef patties held under retail conditions at 1-2°C. The surface of the patties were evaluated for CIE color L* (lightness), a* (redness), and b*

(yellowness) using Chroma Meter CR-400/410 (Konica Minolta Sensing, Inc. Tokyo,

Japan). Three patties per treatment per replication were randomly selected, and three separate readings were obtained from each of the patties for a total of 9 readings. The 9 readings from each treatment sample were averaged and used for statistical analysis.

3.2.5 Texture profile analysis Texture profile analysis was conducted on cooked patties using the Instron

Universal Testing Machine (Model 1011, Instron Corp., Canton, Mass.) following

Bourne’s (1978) method. One 50 mm diameter core from each of four different patties

per treatment per replication was used for texture profile analysis. Each patty core was

compressed twice to 75% of its original height using a 57 mm circular flat disk with full

scale load 500 kilogram (kg) and crosshead speed 100 mm/min. Measurements included

hardness = peak force of first compression (N); springiness = distance (in cm) sample

recovered after first compression or [(distance under curve of second compression divided by distance under curve of first compression) x 0.75 x 2.54 cm (height of patty)]; cohesiveness = total energy area under the curve of second compression divided by the total energy of the first compression dimensionless); gumminess = product of hardness x cohesiveness (N); and chewiness = product of hardness x cohesiveness x springiness (N- cm).

3.2.6 Scanning electron microscopy (SEM) Beef patties with and without starches were evaluated using the SEM instrument

(Jeol-JSM 6301FXV, Akishima, Tokyo, Japan) according to the procedure described by

Beckett and Read (1986). Three beef patties per treatment per replication were selected for SEM analysis. The uncooked patties from these treatments – 1) K6, beef patties with

6% kudzu and 5% water; 2) P6, beef patties 6% potato and 5% water; and 3) NC, negative control with no starch and no water were used for SEM analysis. Treatment samples (~2 gms) were kept overnight in 2.5 % glutaraldehyde in 0.1 M phosphate buffer, pH 7.2 for fixation. These samples were rinsed three times with 0.1 M phosphate 33

buffer for 15 min every time. Thereafter samples were post-fixed in 2% osmium tetroxide

(OsO4) (Sigma Chemical Co., MO, USA) and 0.1 phosphate buffer at room temperature

for 2 h followed by overnight refrigeration at 4°C . The freshly prepared osmium tetroxide is colorless to pale yellow in color, when material (in this case meat samples) reacts, it causes oxidation and samples turn black. The glutaraldehyde is often used to crosslink protein molecules and osmium tetroxide to preserve lipids. All samples exhibited a typical black color change after holding overnight in refrigerator. The black colored samples were rinsed three times with distilled water 15 min each time, and

dehydrated with increasing concentrations of ethanol (35%, 50%, 70%, 95% for 15 min

and 4-6 times at 100% for 15 min) and then critical-point dried (E-300 Critical Point

Dryer; Polaron Equipment, Watford, United Kingdom) where carbon dioxide gas was

used for drying. The dry sections were fractured using a curved sharp scalpel blade (blade

No.10, Bard & Parker, Franklin Lakes, NJ) and fragments were mounted on aluminum

stubs and coated with Au/ Pd gold in vacuum. Images were taken using SEM at 500x,

1.5kx (1000x =1kx) and 3.5kx magnification. Scans were also performed on kudzu and

potato starch granules at 500x and 1.5kx magnification to provide reference images for

these ingredients. Typical micrographs representative of the sample and of similar

fracture planes, were chosen for each of the treatments.

3.2.7 Sensory evaluation

3.2.7.1 Sensory Sample Preparation Beef patties were cooked on a griddle top (Griddle 442A, Toastmaster Inc.,

Booneville, MO). The griddles were preheated to temperature of 176°C for 5mins prior to

placement of beef patties on the griddle. The patties were cooked for 5 min on each side, 34

with an additional 1 minute on each side until an internal temperature of 71°C was

reached as measured by a handheld digital thermometer (Model PT100 RTD, Omega

Engineering Inc, Bridgeport, NJ). This sample preparation was chosen to closely resemble beef patty preparation and consumption in typical consumer homes.

3.2.7.2 Consumer Acceptability Three replications of 60 consumer panelists each were conducted to determine

consumer acceptability of the 6 treatments (K2, K4, K6, P2, P4, P6) and negative control.

Consumers consisted of faculty, staff and students of Mississippi State University solicited through email notices and by word of mouth. The panelists evaluated the

treatment samples in a seven booth sensory room, where lighting, temperature, and

ventilation could be controlled. Panelists received one quarter of each representative beef

patty in a 2-oz plastic lidded container labeled with three-digit random numbers. Panelists

were provided with water, unsalted crackers, and expectorant cups. Each of the treatment

samples were evaluated based on acceptability of flavor, aroma, texture, appearance and

overall acceptability. Evaluations were conducted using a 9-point hedonic scale; 1-dislike

extremely, 2-dislike very much, 3-dislike moderately, 4-dislike slightly, 5-neither like nor

dislike, 6-like slightly, 7-like moderately, 8-like very much and 9-like extremely

(Meilgaard and others 2007). Approved IRB (Institutional Review Board) consent form

and score sheet for consumer acceptability tests are included in Appendix B, C, and D.

3.2.8 Statistical analysis The effects of starch type and starch concentration added to beef patties were

evaluated in the study. The factors of starch type and starch concentration were arranged

in a 2 X 3 factorial arrangement of treatments with batches of beef patties being made

from the combinations of the two starch types (kudzu and potato) and the three levels of

starch concentrations (2%, 4%, and 6%). Three replicates of the experiment were run in

three consecutive weeks. Each batch from a treatment combination (beef logs) was cut

into 1.27 cm beef patties. The responses of pH, cooking yield, expressible moisture,

proximate analysis, texture profile analysis, color evaluation and sensory data was collected on three to five random samples of beef patties from the resultant batch of

ground beef. The experiment was run as a 2x3 factorial arrangement of treatments in a

completely randomized design with subsampling unit’s nested in experimental units,

where:

Factors: A(X1): starch type (kudzu and potato);

B (X2): starch concentration/level (2, 4, and 6%)

Experimental Unit (EU): a batch of beef patty made with one combination of starch type

and starch level

Subsampling Unit (SU): a patty randomly selected from a batch

Response variables (Y): different response variables

The data was analyzed using a two-way cross classification mixed effects model

with SU’s nested in EU’s using PROC GLM from the SAS 9.2. program (SAS 2002).

Model: Yijkl = μ + αi + βj + (αβ)ij + ε (ij)k + δ(ijk)l (3.1)

where αi represents the main effect of starch type (A) i, i = K, P; βj represents the main

effect of starch level (B) j, j = 2, 4, 6; (αβ)ij represents the interaction between starch type

and starch level, ε (ij)k is the experimental error associated with the batches nested in

combinations of starch type and starch level; δ(ijk)l is the observational or subsampling 36

2 2 error associated with patties nested in batches. ε (ij)k ~ N(0, σε ), i.i.d and δ(ijk)l ~ N(0, σ

), i.i.d are assumed to be normally distributed with mean 0 and possess variance

2 2 components σε and σ , respectively. ε (ij)k and δ(ijk)l are assumed to be independent random variables.

Secondly, polynomial regression analysis was used to model moisture, fat content, and protein content as a function of starch level for each starch type. The choice of best regression model was based on maximizing the R-square. Response curves as a function of starch level for each starch type are given in Figure 3.1, Figure 3.2 and Figure

3.3).

[X1]: Starch type

[X2]: Starch concentration/level

Polynomial regression model fit for each response variable as a function of starch level for each starch type;

Model: (3.2)

Thirdly, to assess the effect of kudzu and potato starch on quality characteristics

of beef patties as compared to positive and negative control samples, the data was also

analyzed as a completely randomized design (CRD) with three replications and 8 treatments as outlined in Table 3.3, Table 3.6, Table 3.8, Table 3.10 and Table 3.12. The data was analyzed using a one-way classification mixed effects model with SU’s nested in EU’s using PROC GLM with appropriate LSD multiple comparisons being obtained from LSMEANS (SAS 2002) and a P- value < 0.05 was considered as significant.

(Appendix A)

Model: Yijk = μ + τi + ε (i)j + δ (ij)k (3.3)

th where τi represents the effect of the i treatment, i, i = 1,…8; ε (i)j is the experimental error

associated with the batches nested within the treatments; δ(ij)k is the observational or

2 2 subsampling error. ε (i)j ~ N(0, σε ), i.i.d and δ(ij)k ~ N(0, σ ), i.i.d are assumed to be

2 2 normally distributed with mean 0 and possess variance components σε and σ ,

respectively. ε (i)j and δ (ij)k are assumed to be independent random variables.

Fourthly, Agglomerative hierarchical clustering was used to cluster consumers on

the basis of consumer scores for overall acceptability using XLSTAT version 2011

Software (Addinsoft,. New York, NY). A dendrogram plot was used to determine the

number of clusters that should be used to group consumers. This analysis was performed

on standardized liking scores, considering Euclidean distances and average linkage as

agglomeration criterion. Internal preference mapping was also carried out using a

principal component analysis on the correlation matrix of consumer individual liking data

using XLSTAT version 2011(Addinsoft, New York, NY).

3.3 Results and Discussion Results from this study initially characterized ground beef patties by the change in

physical, chemical, color, textural and consumer responses, as affected by starch type

(kudzu, potato) and starch level (2, 4, and 6%). Starch treated patties were additionally

compared to positive and negative control samples. For certain response variables

(chemical analysis: moisture, fat and protein content) regression models were fit as a

function of starch level for each starch type. The R2 of the model is shown in Table 3.5.

3.3.1 Physical analysis

3.3.1.1 pH The analysis of variance results for the effects of two factors – type of starch (A)

and starch level (B) on the physical properties of beef patties are shown in Table 3.2. The

pH of uncooked patties was significantly (P < 0.05) affected by the starch level.

Averaged over starch type, patties with 6% starch were lower (P<0.05) in pH compared

to 2% and 4% (Table 3.2). This could be due to the increase in solids content coupled

with a decrease in water content in 6% starch samples. Comparing the treatment

combinations to positive and negative control samples; K6, P6, and NC samples were

found to be lower (P < 0.05) in pH value compared to K2, K4, P2 and P4 samples (Table

3.3).

Table 3.2 Analysis of variance on the effects of starch type and starch level on physical properties of beef patties formulated with kudzu and potato starch

Parameters P values A B A x B 0.0001* %(5.7)a pH 0.8578 2 0.9749 4%(5.7)a 6%(5.4)b Cooking Yield (%) 0.5953 0.5807 0.2717 0.0326* 0.02490* 2%(1.3)a Expressible Moisture (%) K(1.3) a 0.5505 4%(1.2)ab P(1.1) b 6%(0.97)b * p < 0.05; A: Starch type K, kudzu, P, potato (mean values)) B : Starch level 2,4, 6%;( mean values) When significant differences existed, observed means were separated with letters a,b. Means in the same column with different letters are significantly different (P<0.05).

3.3.1.2 Cooking yield Type of starch, starch level and type of starch x starch level interaction had no significant effect on the cooking yield of beef patties (Table 3.2). As expected, the

cooking yield of beef patties with kudzu at 4 and 6%, and potato starch at 4 and 6% was higher (P <0.05) than positive and negative control samples. P2 samples were higher (P

<0.05) in cooking yield than PC samples (Table 3.3). No significant difference existed between PC and NC samples with respect to cooking yield. The reason for the increase in cooking yield of beef patties with formulations containing starch and water could be attributed to the fact that starch retained higher moisture and prevented excess loss by evaporation or drip-loss during cooking. The results of this study are in agreement with other research where starch based fat replacers such as- modified corn starch (Khalil

2000), corn starch (Garzon and others 2003), tapioca starch (Nissar and others 2009), sodium alginate (Berry 1997), bambara groundnut (Vigna subterranean L.) (Alakali and others 2010), oat fiber (Pinero and others 2008), potato and corn starch (Aktas and

Genccelep 2006), carrageen gum, modified cassava starch, oat bran (Sampaio and others

2004), and combinations of polydextrose, sugarbeet fiber, oat fiber, potato starch and pea fiber (Troutt and others 1992), were found to increase the cooking yield of meat formulations.

3.3.1.3 Expressible moisture Expressible moisture of cooked patties was significantly (P < 0.05) affected by the starch level (Table 3.2). The two types of starch are significantly different with respect to mean expressible moisture. Averaged over starch type, patties with 6% starch were lower (P<0.05) (0.97) in expressible moisture than 2% (1.3) starch patties (Table 40

3.2). All treatments and PC samples were lower (P <0.05) in expressible moisture (0.91 –

1.9) than NC treatment (4.0) (Table 3.3). Expressible moisture measures loosely bound water in the system. The ability of starch to entrap water and form hydrogen bonds with water called hydration during cooking accounts for the decrease in expressible moisture.

This explains why starch/water combination treatments were lower in expressible moisture compared to NC samples. The results of this study are in agreement with

Motzer and others (1998) and Schilling and others (2001) who observed lower expressible moisture in boneless cured ham with modified food starch compared to control samples with no starch.

Table 3.3 pH (uncooked), cooking yield and expressible moisture (cooked) of beef patties formulated with and without kudzu or potato starch.

Expressible Moisture Treatments1 pH Cooking Yield (%) (%)

2% K+ 5% W 5.7 a 64.9 dc 1.4 b

4% K+ 5% W 5.7 a 72.4 ab 1.4 b

6% K+ 5% W 5.4 c 76.1 a 1.0 b

2% P+ 5% W 5.7 a 67.2 bc 1.2 b

4% P+ 5% W 5.7 a 75.6 a 1.0 b

6% P+ 5% W 5.4 bc 76.7 a 0.91 b

PC (0% starch+ 5% W) 5.6 ab 59.8 d 1.9 b

NC (0% starch+ 0% W) 5.4 bc 61.0 cd 4.0 a P – value 0.0006 <0.0001 0.0460 a, b,c,d Means in the same column with different letters are significantly different (P<0.05). 1 K (kudzu), P (potato), W (water), PC (positive control), NC (negative control) 41

3.3.2 Chemical analysis Chemical properties of ground beef patties as affected by types of starch and starch levels are shown in Table 3.4. Regression analysis was performed to determine the relationship between the chemical properties and the starch levels. Table 3.5 shows the fitted regression equations and the partial t-test results. The coefficient of determination, which for the curves ranged between 56-96 %, indicates how much of the variation in the chemical properties is accounted for by the fitted regression model. Higher percentages are more desirable and give us confidence that the chemical properties are related to starch level through the quadratic regression model. Regressions with low coefficients of determination suggest a response over starch level which is substantially affected by other unidentified factors or which changes little due to starch levels. PC and NC samples were further compared to all treatments in 0.

3.3.2.1 Moisture content Moisture content of uncooked patties from treatments such as K2, K4, K6, and P6 were lower (P < 0.05) in moisture content than PC and NC samples. Cooking caused significant decrease (P<0.05) in moisture content in all samples because of evaporation and drip loss (Table 3.6).

Moisture content of cooked patties was significantly affected by type of starch and starch level but not by the interaction of starch type x starch level. Averaged over starch level, patties with large potato starch granules retained higher (P < 0.05) moisture than samples with smaller kudzu starch granules (Table 3.4). Schilling (2001) and Motzer

(1998) reported that larger surface area of modified food starch held more moisture than smaller surface area of soy concentrates. Averaged over starch type, patties with 6% 42

starch was significantly higher in moisture than 2% and 4% starch patties. The moisture content relationship with starch level was also modeled with a regression model for each starch type. Moisture content showed linear response for both kudzu and potato starch patties, linear in this case meaning that the moisture content of cooked patties increased over starch level (Figure 3.1). No significant difference existed in moisture content among samples of NC, K4, K6, P2 and P4 treatments. PC and P6 samples were higher (P

<0.05) in moisture content than K2, K4, and NC samples. Pinero and others (2008) reported similar result of higher moisture retention in samples with oat fiber and water compared to control-all beef samples.

3.3.2.2 Fat content Fat content of uncooked beef patties ranged from 6.5 to 8.1% for all treatments and controls. All treatment and control samples increased (P <0.05) in fat content after cooking except K6 and P6 (Table 3.6).The increase in fat content in cooked patties was mostly due to the significant moisture loss and ability of starch to prevent fat migration during cooking. K6 and P6 samples were already low in fat content. This coupled with high moisture retention of starch might have prevented patties from increasing in fat content in 6% starch samples. Similar results were reported by Alakali and others (2010),

Kumar and Sharma (2004), and Modi and others (2003 ) who found an increase in fat content in meat patties after cooking.

The fat content of cooked patties was significantly affected by type of starch and starch level but not by type of starch x starch level interaction (Table 3.4). Kudzu starch samples were higher (P<0.05) in fat content than potato starch samples. Averaged over starch type, with every 2% increase in starch level there was a significant decrease in fat 43

content. The regression analysis further described the relationship of fat content to starch

level. The fat content of kudzu starch patties showed significant linear fit while that of

potato starch samples showed significant quadratic fit, meaning the relationship is non-

linear. Figure 3.2 shows that fat content in potato starch decreases non-linearly (Table

3.5, Figure 3.2). K6 and P6 treatment samples were lower (P<0.05) in fat than K2, K4,

P2, PC and NC samples. No difference (P>0.05) existed among K4, P2, P4, PC and NC

treatments with respect to the fat content in cooked patties (Table 3.6).

3.3.2.3 Protein content Protein content of all uncooked patties ranged between 19.2 to 20.3%. P4 and P6

uncooked samples contained lower (P < 0.05) protein content compared to NC samples

(Table 3.6). With addition of various levels of starch, protein content generally decreased

in uncooked patties probably due to the increase in solids content. Cooking caused significant (P < 0.05) increase in protein content of beef patties primarily due to the moisture loss (Table 3.6). Heat treatment generally results in denaturation of the proteins and hydration of the starch (Pinero and others 2008). Similar result was observed by

Alakali and others (2010) who studied the effect of Bambara groundnut (Vigna subterranean L.) seed flour in low fat beef patties and found cooked patties higher in protein content than uncooked patties.

Protein content of cooked patties was significantly affected by type of starch and

starch level. Kudzu samples were higher (P <0.05) in protein content than potato starch

patties. With every 2% increase in starch level, there was a significant (P<0.05) decrease

in protein content. Regression equation for protein content fitted over starch level showed

significant (P< 0.05) linear trend, in this context meaning that the protein content linearly 44

decreased over starch level for both kudzu and potato starch patties (Table 3.5, Figure

3.2). NC samples contained highest (P<0.05) protein content compared to all treatments and positive control. The PC and K2 samples were higher (P<0.05) in protein content compared to K4, K6, P2, P4 and P6 treatments.

Summarizing the results of proximate analysis, cooked patties with kudzu starch were significantly lower in moisture (62.7 vs. 64.4), significantly higher in fat (9.1 vs.

8.3) and significantly higher in protein (26.3 vs. 24.7) content compared to potato starch

samples. As expected, with every 2% increase in starch levels, there was a significant

(P<0.05) decrease in fat content and decrease in protein content in cooked patties. The

differences in morphology, source, and granule size (Suzuki and others 1981;

Achremowicz and Tanner 1996; Srichuwong and others 2005b) might have accounted for

the differences in chemical properties.

Table 3.4 Analysis of variance on the effects of starch type and starch level on chemical properties of beef patties formulated with kudzu and potato starch

Parameters P values A B A x B 0.0013* 0.0004* 2%(62.6)b Moisture (%) K(62.7) b 0.2424 4%(63.3)b P(64.4) a 6%(64.6)a 0.0001* 0.0012* 2%(9.7)a Fat (%) K(9.1) a 0.1093 4%(8.9)b P(8.3) b 6%(7.4)c 0.0001* 0.0001* 2%(27.7)a Protein (%) K(26.3) a 0.3560 4%(25.4)b P(24.7) b 6%(23.4)c * p < 0.05; A: Starch type K, kudzu, P, potato (mean values) B: Starch level 2, 4, 6 %;( mean values) When significant differences existed, observed means were separated with letters a, b. Means in the same column with different letters are significantly different (P<0.05).

Figure 3.1 Fitted curve of moisture content as a function of starch level for kudzu and potato starch cooked patties.

Figure 3.2 Fitted curve of fat content as a function of starch level for kudzu and potato starch cooked patties. .

Figure 3.3 Fitted curve of protein content as a function of starch level for kudzu and potato starch cooked patties.

Table 3.5 Fitted Regression Equations as a function of level of starch on physicochemical properties of beef patties formulated with kudzu or potato starch.

Partial t-test Parameter Fitted Regression Equations P value R2 s Linear Quadratic

K: 59.053 1.308 ∗ 0.0875 ∗ ∗ 0.0098* 0.599 0.6554 K: 60.220 0.608 ∗ 0.0098* - 0.6378 Moisture P: 65.483 1.263 ∗ 0.210 ∗ ∗ 0.0210* 0.085 0.7405 P: 62.672 0.423 ∗ 0.0210* - 0.5565

K: 11.813 0.877 ∗ 0.0408 ∗ ∗ 0.0004* 0.632 0.8528 K: 11.269 0.550 ∗ 0.0004* - 0.8466 Fat P: 8.260 0.855 ∗ 0.1817 ∗ ∗ 0.0576 0.0069* 0.9610

K: 32.217 1.957 ∗ 0.1025 ∗ ∗ <0.0001* 0.295 0.9533 K: 30.850 1.137 ∗ <0.0001* - 0.9431

Protein P: 28.177 0.6575 ∗ 0.0446 ∗ ∗ <0.0001* 0.599 0.9523 P: 28.177 0.6575 ∗ 0.0446 ∗ ∗ <0.0001* - 0.9499

, fitted response; X, the level of starch (2%, 4%, 6%); K, kudzu starch; P, potato starch.; significant at α = 0.05. Proximate composition of uncooked and cooked beef patties formulated with and without kudzu or potato starch.

Table 3.6 Proximate composition of uncooked and cooked beef patties formulated with and without kudzu or potato starch

Treatments1 Moisture (%) Fat (%) Protein (%)

Uncooked Cooked Uncooked Cooked Uncooked Cooked

2% K+ 5% W 68.6 b 61.3 f 8.1 cd 10.2 a 19.8 fgh 28.7 b

4% K+ 5% W 68.4 b 62.8 e 8.0 cde 8.9 b 20.1 fg 26.0 c

6% K+ 5% W 68.2 b 63.8 de 7.7 def 8.0 cde 19.7 fgh 24.2 d

2% P+ 5% W 69.4 ab 63.8 de 7.8 def 9.2 b 20.1 fg 26.7 c

4% P+ 5% W 69.0 ab 63.8 de 7.1 fg 8.7 bc 19.4 gh 24.8 d

6% P+ 5% W 68.5 b 65.5 c 6.5 g 6.9 g 19.2 h 22.6 e PC (0% starch+ 70.5 a 64.9 cd 7.2 efg 9.2 b 19.6 fgh 28.7 b 5% W) NC (0% starch+ 69.5 a 62.8 e 7.9 de 9.5 ab 20.3 f 31.2 a 0% W) P – value <0.0001 <0.0001 <0.0001 a, b,c,d,e,f,g,h Means in the same column with different letters are significantly different (P<0.05). Means in the same row of uncooked and cooked patties with different letters are significantly different. 1 K (kudzu), P (potato), W (water), PC (positive control), NC (negative control)

3.3.3 Color evaluation

CIE L*, a* and b* evaluations were performed on uncooked patties. The effect of two factors- type of starch and starch level on color parameters is presented in Table 3.7.

Color characteristics of treatment samples are compared to controls in Table 3.8.

CIE L* value was affected (P<0.05) by type of starch x starch level interaction.

Results indicated that kudzu starch treatments (K2, K4, K6) were lighter (P < 0.05) than potato starch treatments (P2, P4, P6), and potato starch sample at 4% and 6% were lighter

(P < 0.05) than PC and NC samples. CIE L* value of K6 treatment was higher (P <0.05)

than K2 and K4. No difference (P >0.05) existed among potato starch treatments. PC and

NC samples were not different (P>0.05) with respect to CIE L* values. The white to light

yellow color of starch flours might have contributed to the higher CIE L* values in

treatment samples compared to PC and NC samples.

Table 3.7 Analysis of variance on the effects of starch type and starch level on color parameters of uncooked beef patties formulated with kudzu and potato starch

Parameters P values A B A x B CIE L* 0.0415* CIE a* 0.8677 0.6760 0.9727 CIE b* 0.0537 0.5517 0.7105 * p < 0.05; A: Starch type K, kudzu, P, potato (mean values) B: Starch level 2,4,6% ( mean values) When significant differences existed, observed means were separated with letters a, b. Means in the same column with different letters are significantly different (P<0.05).

CIE a* (redness) or CIE b* (yellowness) values of patties were not significantly

affected by type of starch, starch level and type of starch x starch level interaction (Table

3.7). No difference (P > 0.05) existed among treatments, PC and NC samples with respect

to CIE a* values. The redness values of patties ranged from 13.2 to 14.4 (Table 3.8). CIE

b* values of K2 (6.3), K4 (6.4), K6 (8.0) treatments were higher (yellower) (P < 0.05)

than PC (4.7), NC (4.6) and P2 (5.0) samples (Table 3.8).

Overall considering the color parameters, kudzu treatments (K2, K4, and K6)

were lighter (P<0.05) and yellower (P<0.05) than PC and NC samples. Potato starch

samples were lighter (P<0.05) than NC samples. Our results are in agreement with

Brewer and others (1992) who reported higher L* and b* values in beef patties containing

carrageenan starch compared to the control all- beef patties.

Table 3.8 CIE L*, a*, b* color mean values of uncooked beef patties formulated with and without kudzu or potato starch.

Treatments1 L* a* b*

2% K+ 5% W 49.5 b 13.5 6.3 b

4% K+ 5% W 50.0 b 14.2 6.4 b

6% K+ 5% W 53.0 a 13.9 8.0 a

2% P+ 5% W 47.2 cd 13.4 5.0 c

4% P+ 5% W 47.5 c 14.4 5.3 bc

6% P+ 5% W 47.8 c 14.2 5.4 bc

PC (0% starch+ 5% W) 45.9 de 13.2 4.7 c

NC (0% starch+ 0% W) 45.1 e 13.6 4.6 c

P – value <0.0001 0.8102 0.0004 a, b,c,d,e Means in the same column with different letters are significantly different 1 K (kudzu), P (potato), W (water), PC (positive control), NC (negative control)

3.3.4 Texture profile analysis Table 3.9 shows the analysis of variance results for the effects of two factors – type of starch and starch level on the textural attributes of cooked beef patties. The mean scores of hardness, springiness, cohesiveness, chewiness and gumminess of all treatments

(K2, K4, K6, P2, P4, and P6) are compared to controls (PC, NC) in Table 3.10.

Hardness of cooked patties was significantly affected by type of starch with kudzu starch being significantly harder than potato starch (Table 3.7). No significant (P >0.05)

difference existed among treatments K2, K4, K6, P2, P4, P6, PC, and NC with respect to

the hardness of beef patties (Table 3.10). However, averaged over the starch levels potato

(9.3N) starch beef patties were found to be lower (P<0.05) in hardness compared to

kudzu patties (6.9N). This could be due to the fact that potato starch granules were larger

in size and retained higher moisture than kudzu starch granules. Khalil and others (2000)

reported similar results where they found beef patties with higher moisture, lower in

hardness.

Table 3.9 Analysis of variance on the effects of starch type and starch level on textural properties of beef patties formulated with kudzu and potato starch

Parameters P values A B A x B 0.0397* Hardness (N) K (9.3) a 0.8295 0.9774 P( 6.9) b Springiness (cm) 0.2085 0.2567 0.1169 Cohesiveness 0.2680 0.2344 0.1172 Chewiness (Ncm) 0.0100* 0.0100* Gumminess (N) K (3.7) a 0.4867 0.5034 P(1.9) b * p < 0.05; A: Starch type K, kudzu, P, potato (mean values) B: Starch level 2,4,6%;( mean values) When significant differences existed, observed means were separated with letters a, b. Means in the same column with different letters are significantly different (P<0.05).

Table 3.10 Texture profile analysis of cooked beef patties formulated with and without kudzu or potato starch.

Hardness Springiness Chewiness Gumminess Treatments1 Cohesiveness (N) (cm) (Ncm) (N) 2% K+ 5% W 9.7 1.0 ab 0.44 bc 4.3 a 4.3 a

4% K+ 5% W 8.9 0.74 bc 0.31 de 1.9 bc 2.8 abcd

6% K+ 5% W 9.4 1.1 ab 0.56 a 4.3 a 4.0 ab

2% P+ 5% W 7.4 1.0 abc 0.35 cd 2.2 b 2.1 bcd

4% P+ 5% W 6.7 0.82 abc 0.28 de 1.6 bc 1.9 cd

6% P+ 5% W 6.6 0.60 c 0.24 e 1.0 c 1.6 d PC (0% starch+ 8.5 1.2 a 0.49 abc 4.5 a 3.8 abc 5% W) NC (0% starch+ 8.4 1.2 a 0.45 ab 4.8 a 3.9 ab 0% W) P – value 0.5352 0.0318 0.0215 <0.0001 0.0262

a, b,c,d,e Means in the same column with different letters are significantly different (P<0.05). 1 K (kudzu), P (potato), W (water), PC (positive control), NC (negative control) N= Newton, cm=centimeter, Ncm=Newton centimeter

Springiness of cooked patties was not significantly (P >0.05) affected by type of

starch x starch level interaction, type of starch or starch level. K4 and P6 samples were

found to be lower (P<0.05) in springiness than NC and PC samples (Table 3.10). The

well-structured continuous meat matrix made with muscle fibers and holes of evaporated

water might have allowed PC and NC samples to be springier than potato and kudzu

samples where starch granules were embedded within the protein matrix of beef patties.

Cohesiveness of cooked beef patties was not significantly affected by type of starch x starch level interaction, type of starch or starch level (Table 3.9). K4, P4, and P6

samples were found to be lower (P <0.05) in cohesiveness than K2, K6, PC and NC

patties. No difference (P > 0.05) existed among K2, PC and NC treatments (Table 3.10).

Khalil and others (2000) observed beef patties with corn starch lower in cohesiveness

compared to control patties.

Chewiness of cooked patties was significantly affected by type of starch x starch

level interaction (Table 3.9). Chewiness of K4, P2, P4 and P6 samples were lower (P <

0.05) in chewiness than K2, K6, PC and NC samples. No difference existed among K2,

K6, PC and NC samples with respect to the chewiness. It appears that kudzu starch

samples at 2% and 6% were chewier like PC and NC while at 4% the chewiness was

closer to the potato starch samples (Table 3.10).

Gumminess was affected (P<0.05) by type of starch in beef patties. Averaged

over starch levels, potato (1.9 N) starch samples were lower (P<0.05) in gumminess than

kudzu starch samples (3.7 N). K2, K6, PC and NC samples was greater (P <0.05) in

gumminess compared to P6 samples. There was no difference (P > 0.05) in gumminess

values among samples of K2, K4, K6, PC and NC treatments.

Overall summarizing the results of texture profile analysis, on an average potato

starch samples (2, 4, and 6%) were found to be lower (P<0.05) in hardness and

gumminess compared to kudzu patties. The results showed that samples from treatments

K4, P6 were lower (P<0.05) in springiness, K4, P4, P6 were lower (P<0.05) in

cohesiveness and K4, P2, P4, P6 were lower (P<0.05) in chewiness compared to PC and

NC samples. It appears that kudzu samples at 4% were similar in texture properties

(springiness, cohesiveness, chewiness) to potato patties, while at 2 and 6% texture of kudzu starch samples were more similar to PC and NC samples. Based on the results,

kudzu starch could be well suited for products where cooking yield is intended to

increase, fat content to decrease, while keeping the overall texture similar to control

samples. The optimization of starch and water level is a potential area of exploration.

3.3.5 Scanning electron microscopy The scan images of patties with and without potato starch or kudzu starch at 6% starch and 5% water and NC (negative control with no starch/ no water), are shown in

Figure 3.4 – 3.8. The SEM images of uncooked beef patties of NC are shown in. The NC images (Figure 3.4) illustrate the disrupted muscle fibers, fiber bundles and fat globules dispersed within protein network of beef patties in a random structure. A typical hamburger is made by mincing, blending of meat fibers and fiber bundles, which are randomly distributed compared to the well-defined anisotropic structure of the whole muscle (Tornberg 2005). The negative control beef patties contained 70% water, 20% protein, and 8% fat (Table 3.6). The fat globules can be seen in Figure 3.4. The open

spaces between the muscle cells indicate the presence of water, which was sublimed

during sample preparation for SEM. The SEM images of meat structure in a beef patties

in this study were similar to the microscopy images of beef patty samples and beef

sausage shown by Ahmed and others (2009) and Morin and others (2004).

The microscopy images of patties with 6% potato starch or kudzu starch are

shown in Figure 3.5 and Figure 3.6. Potato starch showed a large rounded and typical

round-shaped granule (Figure 3.5). Spherical, hemispherical and polygonal shaped

granules can be seen for kudzu starch (Figure 3.6). SEM images showed that kudzu

starch granules were smaller than potato starch. Kudzu starch has been reported to be

short, sharp edged and angular compared to corn and lotus starch (Geng and others 2007). 55

Similar to results reported by Hung and Morita (2007) and Srichuwong and others

(2005b), kudzu starch granules in this study were found to be both spherical and polygonal. Achremowicz and others (1996) observed 80 % of the kudzu starch granules’ average diameter less than 32 microns. Potato starch granule size has been reported to range between 50-100microns- an average of 40 microns and shape as mostly oval or spherical (Anonymous 2011). The difference in granule morphology may be attributed to the biological origin, biochemistry of the amyloplast and physiology of the plant (Sandhu and others 2004).

SEM images of uncooked beef patties of P6 and K6 treatments are shown in

Figure 3.7 and Figure 3.8, respectively. The potato granules interfere with the protein network of meat matrix, weakening the compactness of intact meat by partially embedding in the spaces within muscle fibers. The microscopy images of beef patties with K6 treatment illustrate the denser matrix with smaller granules very intact b/w muscle fibers (Figure 3.8). This indicates that kudzu granules did not disturb the protein network as much as potato starch granules and thus improved water binding properties of beef patties at the same time maintaining compactness of intact muscle. Kudzu starch patties were higher in hardness, cohesiveness and gumminess compared to potato starch patties. SEM images on cooked patties were not performed in this study and could be assessed in future.

Figure 3.4 Scanning electron microscopy images of uncooked beef patties with no starch and no water (Negative Control)

(a) at 500x and (b) at 1.5kx (c) at 3.5kx magnifications . F, fat globule and M, muscle fiber.

Figure 3.4 (Continued)

Figure 3.5 Scanning electron microscopy images of potato starch

(a) at 500x, and (b) at 1.5kx magnifications. P, potato starch .

Figure 3.6 Scanning electron microscopy images of kudzu starch

(a) at 500x and (b) at 1.5kx magnifications. K, kudzu starch.

Figure 3.7 Scanning electron microscopy images of uncooked beef patties with 6% potato starch and 5% water (P6)

(a) at 500x, (b) at 1.5kx and (c) at 3.5kx magnifications. F, fat globule and M, muscle fiber, P, potato starch.

Figure 3.7 (Continued)

Figure 3.8 Scanning electron microscopy images of uncooked beef patties with 6% kudzu starch and 5% water (K6)

(a) at 500x, (b) at 1.5kx, and (c) at 3.5kx magnifications. F, fat globule and M, muscle fiber, K, kudzu starch.

Figure 3.8 (Continued)

3.3.6 Sensory evaluation / Consumer acceptability Panelists evaluated appearance, texture, flavor and overall acceptability of beef patties formulated with and without starch/ water combination treatments on 9 point hedonic scale (1= dislike extremely, 5= neither like nor dislike, 9= like extremely). The negative control (NC), K2, K4, K6, P2, P4, and P6 treatments were chosen, but not PC treatment, for sensory analysis to decrease the number of samples for panelists considering their fatigue, and these are the samples that are most likely to be sold commercially (Table 3.12). Results indicated that the two factors- starch type and starch level had no significant effect on the appearance, aroma, texture and flavor of beef patties. ’Overall acceptability’ was affected by type of starch (Table 3.11). No significant

(P >0.05) difference existed among treatments K2, K4, K6, P2, P4, P6, PC, and NC with respect to appearance, aroma, texture, flavor and overall acceptability of beef patties

(Table 3.12), however, averaged over the starch levels potato (5.7) starch beef patties were found to be higher (P<0.05) in ‘overall acceptability’ attribute compared to kudzu patties (5.38) (Table 3.11). A difference of 0.3 on 9 point hedonic scale (1= dislike extremely; 9 = like extremely) at significance level P= 0.0431 is of relatively slight importance. The overall liking score of all samples ranged between 5 “neither like nor dislike” and 6 “like slightly” (Table 3.12). One probable reason for all samples to score relatively lower hedonic scores could be the sampling method where one quarter of a beef patty was provided to panelists compared to practical beef patty consumption style with bread/lettuce/tomato/spices in sandwich style. Choi and others (2012) reported similar result of overall liking scores using 9pt. hedonic scale for control pork patties made with

20% back fat (4.3) and treatments (5.2 - 6.5) with back fat and fat replacer made from the porcine longissimus dorsi muscle at different levels. Yi and others (2012) used a 7-point hedonic scale (1 = dislike extremely; 7 = like extremely) in place of 9 pt. scale and reported overall liking scores of beef patties made with 0, 1, 3, and 5% glutinous rice flour as 3.3, 4.8, 4.3 and 4.5, respectively. There are various studies where starch based fat replacers have been reported to improve texture and mouthfeel and increase juiciness and tenderness of meat products (Aktas and Genccelep 2006; Berry 1997; Berry and

Wergin 1993; Garzon and others 2003; Nissar and others 2009; Sampaio and others

2004; Shih and Daigle 2003; Warner and others 2001; Serdaroglu 2006; Troutt and others

1992).

Table 3.11 Analysis of variance on the effects of starch type and starch level on consumer acceptability of beef patties formulated with kudzu and potato starch

Parameters P values A B A x B Appearance 0.1860 0.4498 0.5806 Aroma 0.5186 0.4967 0.9061 Texture 0.1420 0.4791 0.7361 Flavor 0.0927 0.7091 0.4229 0.0431* Overall Acceptability K(5.4) b 0.9071 0.6306 P (5.7) a * P < 0.05; A: Starch type K, kudzu, P, potato (mean values). B: Starch level 2, 4, 6 %; ( mean values). When significant differences existed, observed means were separated with letters a, b. Means in the same column with different letters are significantly different (P<0.05).

Since consumers vary significantly in their perception of acceptability, cluster analysis was performed to identify groups of consumers with different preference patterns. The panelists (n= 148) were grouped into 4 clusters based on the overall acceptability ratings and preference of treatments and negative control samples (Table

3.13). Cluster 1 (36% of panelists) was the largest consumer group. These consumers liked all treatments, but preferred (P <0.05) P2, P4, P6 and K6 over K2, K4, and NC samples. Cluster 2 (26% of panelists) preferred (P<0.05) K2, K4, P2, over K6 and P6 samples. Cluster 3 (17% of panelists) preferred (P <0.05) P6, P2, and NC over K2, K4,

K6 and P4 samples. Cluster 4 (21% of panelists) had the highest degree of liking (7.3) where panelists preferred (P < 0.05) K4, K6, P6 over K2, P2, P4 and NC samples. The hierarchical cluster analysis did not show a clear segmentation in consumers’ responses.

This suggests that panelists liking was heterogeneous and the sample caused different

affective reactions in consumers. None of the clusters showed any dislike for kudzu

starch samples as compared to potato starch samples. Panelists (83%) from cluster 1, 2, and 4 preferred kudzu and/or potato starch/water combination beef patties over negative

control samples (Table 3.13).

Table 3.12 Consumer acceptability of cooked beef patties formulated with and without kudzu or potato starch 1, 2, 3

Overall Treatments1 Appearance Aroma Texture Flavor Acceptability 2% K+ 5% W 6.0 6.2 5.6 5.3 5.4

4% K+ 5% W 6.4 6.5 5.4 5.4 5.5

6% K+ 5% W 6.0 6.1 5.5 5.1 5.3

2% P+ 5% W 6.2 6.3 6.0 5.7 5.8

4% P+ 5% W 6.4 6.6 5.7 5.4 5.6

6% P+ 5% W 6.5 6.4 5.6 5.6 5.7 NC (0% starch+ 0% 6.0 6.2 4.9 5.0 5.1 W) P – value 0.4292 0.8114 0.0828 0.2080 0.2111 a, b Means in the same column with different letters are significantly different (P<0.05). 1 K (kudzu), P (potato), W (water), NC (negative control) 2Hedonic scale was based on a 9-point scale: 1= dislike extremely, 5= neither like nor dislike 9= like extremely. 3n= 138

Table 3.13 Agglomerative Hierarchical Clusters (AHC) of beef patties with and without kudzu or potato starch based on consumer overall liking 1, 2

Consumer Panelists, n Treatments1 group (%) NC (0% 2% K+ 5% 4% K+ 6% K+ 5% 2% P+ 5% 4% P+ 6% P+ 5% starch+ 0% W 5% W W W 5% W W W) 1 57 (36%) 5.14 c 4.79 c 5.86 b 6.25 ab 6.61 a 6.09 ab 4.86 c 2 42 (26%) 6.38 a 5.83 ab 4.8 cd 5.81 ab 5.33 bc 4.381 d 5.22 bc 3 27 (17%) 4.74 cde 4.81 cd 3.89 e 5.70ab 4.04 de 6.52 a 5.31 bc 4 33 (21%) 4.88 cd 7.33 a 6.42 b 4.97 cd 5.33 c 6.66 ab 4.34 d a, b Means in the same row with different letters are significantly different (P< 0.05). 1 K (kudzu), P (Potato), W (water), NC (Negative control) 2Hedonic scale was based on a 9-point scale: 1= dislike extremely, 5= neither like nor dislike 9= like extremely

Figure 3.9 Agglomerative Hierarchical Clusters- C1, C2, C3, C4 illustrating group of consumers (n=159) with different preference patterns based on overall liking scores of beef patties with and without kudzu or potato starch.

In order to develop a deeper understanding of each consumers’ overall liking scores, internal preference mapping was carried out using principal component analysis 68

on consumers’ individual overall liking scores of seven samples. Internal preference map of consumers’ overall liking scores for the K2, K4, K6, P2, P4, P6 and NC samples is shown in Figure 3.10 a, b. Figure 3.10 a shows consumers’ representation and Figure

3.10 b shows samples’ representation. The first two principal components explained

50.07% of the variability of the experimental data. The direction of each vector represents the direction of increasing liking for each individual consumer. The length of the vector is directly proportional to the amount of variance explained by the first two preference dimensions for each consumer (Figure 3.10). Samples in the particular quadrant correspond with the consumers’ representation of that quadrant. As shown, consumers’ likings were located all over the four quadrants and not at one location, revealing that consumers’ preferences were highly heterogeneous. The autonomous victory of a sample with highest overall liking score was absent, in agreement with the fact that all samples received similar (+/- 0.1- 0.7) mean overall liking score (Table 3.12).

These results suggest that an overwhelming majority of consumers did not have any dislike for the kudzu beef patties and liked the kudzu beef patties at least as well as patties manufactured with potato starch.

Variables (axes F1 and F2: 50.07 %) Observations (axes F1 and F2: 50.07 %)

1 (a) (b) 93 15 0.75 41 87 K4 14 14 7 85 14 8 0.5 84 10 7 50 10 1222 3 112 13 5 15 7 14 0 10 2 0.25 40 81 63 76 10 0 69 7 5 516054 7082 K6 0 NC 94 3 10 9 14 9 14 6 52 0 F2 (20.01 %)

15 3 %) F2(20.01 -0.25 1428 2 4846 10 5 79 12 9 12 0 31 1212 6 7 K2 P6 80 9813 6 4210 151 12 2 154158 30 4 11512 4 -0.5 26 111

70 78 72 -5 43 53 88 9 118 13 0 10 6 12 1 P2 -0.75 68 P4 66 14 4 10 4 21 116 -10 -1 -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 - 15 - 10 - 5 0 5 10 15 2 0 F1 (30.06 %) F1 (30.06 %)

Figure 3.10 Internal preference map of consumers’ liking scores of the beef patties with and without kudzu or potato starch

(a) consumers’ representation and (b) samples’ representation, K2, 2% kudzu and 5%water; K4, 4% kudzu and 5%water; K6, 6% kudzu and 5%water; P2, 2% potato and 5%water; P4, 4% kudzu and 5%water; P6, 6 % potato and 5%water.

3.4 Conclusions This study compared the effects of kudzu and potato starch at three levels -2, 4,

and 6% in beef patties with respect to physical properties (pH, cooking yield, expressible

moisture), chemical properties (protein, fat, moisture), texture profile analysis (hardness, springiness, cohesiveness, chewiness and gumminess), color analysis (L*, a*, b*),

scanning electron microscope images, and consumer acceptability (appearance, aroma,

flavor, texture and overall acceptability). A positive control was made with no starch and

5% water, and a negative control was made with no starch and no water. Averaged over

starch levels, potato starch beef patties were found to be higher (P <0.05) in moisture,

lower (P <0.05) in fat and lower (P <0.05) in protein as compared to kudzu starch beef

patties. Scanned electron microscope images indicated that kudzu starch granules were

smaller in size than potato starch. Smaller granule size indicates that kudzu starch

granules did not disturb the protein network as much as potato starch granules and thus

improved the water binding properties of beef patties and at the same time maintaining

the compactness of muscle fibers. Texture Profile Analysis showed beef patties with

kudzu starch to be significantly harder and gummier than potato starch patties. The

increase in starch level caused increase in total moisture and decrease in expressible

moisture, fat content, and protein content. As expected, starch levels at 4% and 6% were

significantly higher in cooking yield than PC and NC samples. Compared to beef patties

with no starch and no water, certain samples with starches were lower in protein, lower in

fat and higher in moisture content. Uncooked beef patties with starches were lighter

(P<0.05) (whiter) in color than PC and NC samples. Consumer acceptability tests showed

no significant differences among treatment or control samples for appearance, aroma,

flavor, and texture liking. However, averaged over starch levels, potato starch (5.7) scored higher (P <0.05) in overall liking scores compared to kudzu (5.3) starch patties.

The overall liking score of all samples ranged between 5 “neither like nor dislike” and 6

“like slightly”. A difference of “0.3” in value on a 9 point hedonic scale (1=dislike extremely; 9= like extremely) in overall liking score between potato and kudzu samples

at significance level P= 0.0431 is of relatively little practical importance.

This research illustrated the physicochemical properties of kudzu starch compared

to conventionally used potato starch in beef patties. The study demonstrated that kudzu

starch has the potential for use as an alternative gluten free starch in food products and

overall, consumer responses to the patties made with kudzu starch were equally positive

to patties formulated with potato starch. However more research is required to confirm

acceptance performance. The study indicates the inclusion of kudzu starch in future

studies like- fat replacers or texturizers, optimization of starch level with water,

utilization of modified form of starch (fat composites, prior-acid or base treated) and

application suitability (yogurt, meat, soup, crackers and others) could be investigated.

CHAPTER IV

QUALITATIVE AND QUANTITATIVE ESTIMATION OF ISOFLAVONES IN

KUDZU ROOT EXTRACT AND BEEF PATTIES CONTAINING VARIOUS

LEVELS OF KUDZU ROOT EXTRACT USING HPLC

4.1 Introduction Phytoestrogens comprise a variety of structurally diverse chemicals, with flavonoids as their largest group. Isoflavones (dietary phytoestrogens) are a class of

flavonoids that are available in the seeds and other parts of various plant species mostly

belonging to Leguminosae family. Isoflavones have a similar structure to β-estradiol of

mammals which allows it to reduce or activate estrogenic activity in the body by reacting

with the estrogen receptors on cells. (Choi and Ji 2005; Delmonte and Rader 2006).

Kudzu (Pueraria lobata) native to Japan and China (called “Gegen”) has been

traditionally used to relieve fever, promote salivation, and relieve thirst, stop diarrhea,

cure cold and relieve wrist and shoulder stiffness (Chinese Pharmacopoeia Committee

2005; Sibao and others 2007). Kudzu root has been studied to contain high amounts of

isoflavones- puerarin, diadzin, diadzein, genistin, genistein, formononetin, and their

derivatives (Delmonte and Rader 2006; Wu and others 2011; Chen and others 2001;

Benlhabib and others 2004). Studies have reported kudzu root to have antiproliferative

effects on cancer cells, hypoglycemic effect, antispasmodic effect, and antioxidative

properties. It also improves blood circulation, prevents heart disease, and suppresses 73

voluntary alcohol intake (Jun and others 2003; Keung 2002; Choi and Ji 2005; Johnson and Loo 2000; Mazur and others 1998; Patel and others 2001). Kudzu root has been reported to relieve hypertension, migraine headaches, and acute deafness, all of which may result from the improvement of cerebral circulation. It was reported that the injection

puerarin into the carotid artery dilates cerebral blood flow and thus improves cerebral

circulation (Qicheng 1980). The crude extract has proved to be effective as an antidipsotropic agent (alcohol intake suppressive) for the management of alcohol abuse.

According to the study by Keung and Vallee (1993), the daily intraperitoneal administration of a crude extract of kudzu roots (at a dose of 1.5g/kg per day) significantly suppressed ethanol intake by more than 50% in alcohol-preferring Syrian golden hamsters given a choice between alcohol solution and water. Diadzein and diadzin were reported as the active isoflavones that accounted for the effect. Diadzein and diadzin suppressed alcohol intake in these animals at doses of 150-230mg/kg per day, respectively (Fang and others 2005; Lai and Tang 1989; Keung 2002). Kudzu root and its extracts are commercially available as dietary supplements in health food stores and on internet websites (Prasain and others 2003; Delmonte and Rader 2006). Dietary supplements are not regulated by the Food and Drug Administration. However, before the agency allows a new food or drug on the market the manufacturer must submit proof that the product is safe (Benlhabib and others 2004).

Commercial dietary isoflavone supplements often contain soy, red clover or kudzu extracts, and in most cases a combination of extracts from different sources

(Delmonte and Rader 2006). Each plant has a characteristic isoflavone profile. Puerarin, which accounts for about 80% of isoflavones in kudzu, is not found in soy- based

products (Nakamura and others 2000). Kudzu root (Pueraria radix) contains 10 times more isoflavones as compared to soybeans (amounting to 2 g/ 100 g dry weight) (Choi and Ji 2005).

The food industry and researchers strive to meet consumer demands and focus on

utilization of non-culinary/nutraceutical herbs or extracts that may potentially be a

functional ingredient. Researchers have used ginger, cinnamon bark, licorice root,

rehmania root, peony root and angelica root to mask flavor in goat meat (Kim and others

1993). Ethanol extracts of white peony, red peony, sappanwood, mountain peony and

rosemary extract have been used in ground beef to reduce lipid oxidation (Han and Rhee

2005). Rosemary extract is capable of precluding lipid oxidation and irradiation-induced

quality changes in ready-to-eat turkey bologna. Bloukas and others(1999) also observed

increased overall acceptability of frankfurters formulated with extract of betanin (extract

from beet juice) and paprika juice. Kudzu root with its touted nutraceutical benefits

(Johnson and Loo 2000; Patel and others 2001; Mazur and others 1998) has potential to

be an important ingredient in value-added food products.

The thermal stability of soy based isoflavones has been studied in detail and it is

generally accepted that isoflavones are not destroyed by cooking. However quantitatively

isoflavones loss could be due to the leaching of isoflavones in cooking water (Uzzan and

Labuza 2004). Conversely, other researchers have observed that isoflavones degrade at

different temperatures in food systems and in the purified forms (Eisen and others 2003;

Uzzan and Labuza 2004; Xu and others 2001). Xu and others studied the changes in

daidzin, genistin, and glycitin in model systems at 95 to 215°C and found no significant

changes up to 110°C, but isoflavone glucosides started to degrade significantly after

135°C. Diadzin, genistin, and glycitin decreased to about 65, 74, and 98% of initial

concentrations, respectively, after 3 min of heating at 215°C (Xu and others 2001). Total

isoflavone concentration was shown to significantly decrease with higher temperature

processing such as roasting and explosive puffing. Eisen and others (2003) reported that

genistin in soymilk decreased at 15-37°C and both genistin and diadzin decreased at 70-

90°C during storage. Baik and others (2005) investigated the thermal stability of kudzu

root isoflavones and variations in the amount of isoflavones in kudzu root extract by

heating at 80, 100, 121, 140, 165 and 180°C for 90 min and found isoflavones diadzin

and genistin to be stable up to 121°C but started to degrade thereafter. Kudzu root extract

has not been studied in meat products such as beef patties. The aim of this study was to

prepare a root tincture from kudzu plants grown in the southeastern parts of the United

States, to identify and quantify the isoflavones in kudzu root crude extract, and to

determine the thermal stability of isoflavones in beef patties.

4.2 Materials and Methods

4.2.1 Reagents Acetonitrile, dimethylsulphoxide (DMSO), acetic acid, hydrochloric acid and

ammonium hydroxide were obtained from J.T. Baker (Phillipsburg, NJ). Isoflavones

were purchased from several suppliers. Diadzein, diadzin, genistein and genistin were

purchased from LC laboratories (Woburn, MA). Glycitein, formononetin, puerarin were

purchased from ICC Inofine (Hillsborough, NJ). Biochanin A and ononin were purchased

from Sigma (St. Louis, MO). Calycosin was purchased from Chromadex (Santa Ana,

CA).

4.2.2 Preparation of stock and calibration solutions Each isoflavone stock solution was prepared by dissolving with DMSO

(Delmonte and Rader 2006) in volumetric flasks. Five milligrams each of puerarin,

diadzin, genistein, genistin, formononetin and biochanin A was dissolved in 5 ml of

DMSO and 10 mgs of diadzein, and glycitein were dissolved in 10 ml of DMSO. One

milligram each of ononin and calycosin were dissolved in 5 ml of DMSO in separate

volumetric flasks. The different amounts of isoflavones in stock solution were due to the

limited availability of reference materials. Stock solutions were stored at -20°C before

being used.

The standard solution was prepared by mixing 1 ml of each of the 10 isoflavones

in a volumetric flask and diluting to 20 ml with DMSO. The prepared standard solution

was filtered through a 0.45μm PTFE (polytetrafluoroethylene) membrane before HPLC

analysis. The calibration curve for each isoflavones was obtained by adjusting the

injecting volume to 5, 10, 20, 30, and 40μl of standard solution accurately measured with gas tight volumetric syringes of HPLC (High Performance Liquid Chromatography). In the range of isoflavones concentrations investigated, the regression equation y=ax of each analyte showed a least square fit (R2) greater than 0.999 (data not shown).

4.2.3 Plant sample preparation Kudzu root extract was prepared according to the procedure described by Jun and

others (2003) Kudzu root extract was prepared and supplied by Wild Pantry, Mother

Nature’s Super Store (Tellico Plains, TN). The kudzu root (Pueraria lobata) was dug in

the month of September from the area in Monroe County, TN, between Tellico Plains,

TN and Madisonville, TN. The kudzu root was washed, cleaned and chopped into small 77

pieces. The 8 oz (226.80 gm) of chopped kudzu root was kept in a glass container with 16 oz (453.60 gm) of 40% (80 proof) ethanol. The lid was closed tightly to prevent alcohol evaporation and the glass container was stored in a cool dry place (cabinet) where direct sunlight could not reach. The container was turned upside down and left turned over until the next turning twice in a day with a gap of at least 7-8 hours in between the turnings.

After two weeks of storage, the extract was drained off and decanted into an amber bottle and shipped to the Department of Food Science at Mississippi State University. One milliliter of kudzu root crude extract was also diluted to 5 ml, and 10 ml in volumetric flasks with DMSO to obtain crude extract: 5 and crude extract: 10 samples, respectively.

Kudzu root crude extract, crude extract: 5; and crude extract: 10 diluted samples were analyzed and compared to a standard solution prepared with reference materials. The crude kudzu root extract samples were filtered through a 0.45μm PTFE membrane before

HPLC analysis.

4.2.4 HPLC analysis HPLC was performed at the Mississippi State Chemical Laboratory using Waters

2695 Separation Module (Waters Association, Milford, MA) equipped with Waters 2996 photodiode array detector (PDA) according to the procedure of Delmonte and Rader

(2006) . The reversed phase column used was Alltech® Alltima™ HP C18 HL 5μm, 250 x 4.6 mm, 1.0 ml/min. The column temperature was kept constant at 40°C. The UV wavelength was set at 203 nm. Samples were eluted at 1ml/min with a linear gradient of

0.1% MeCOOH in water (A) and 0.1% MeCOOH in MeCN (B). The gradient was as follows: from 5% B to 20% B over 50 min, then from 20% B to 40% B over 40 min. At the end of run, the column was washed for 10 min with 70% B before returning to the 78

original conditions. The complete run was 100 min, and the column was re-equilibrated for 20 min before the next run. The injection volume was fixed at 10μl for all the

analyses. Each sample analysis was processed by Waters Empower Software 2002

equipped with a chromatographic pattern matching tool.

4.2.5 Validation of HPLC method, identification of peaks and calculations of isoflavones content

The chromatographic pattern match processing method parameters used for the comparisons of samples were optimized as follows. Replicate injections of identical samples were carried out and used to develop method parameters, namely scan start and stop times, peak width, alignment interval, retention time search limit, detection threshold, response value and percent peak height. The scan start time and stop time was

0 and 100.6 min. The response value and percent peak height were 0.0005 and 4.3%, respectively. After these parameters were optimized, the same pattern match processing method was then applied to all the samples analyzed.

Peaks in the samples were identified by an automatic procedure based on the comparison of relative retention times (RRT) using the method developed by Empower software. The amount of isoflavones present in the samples was automatically obtained from HPLC, based on the calibration curve of individual isoflavones. The calibration

curves were made from the known amounts of isoflavones in the standard solution. The data obtained from HPLC was then back calculated (details explained in next section) to obtain the isoflavone content in the samples using dilutions of sample preparation.

4.2.6 Calculations: For kudzu root extract:

For example, data obtained from HPLC in kudzu root crude extract without dilution is calculated accordingly:

Amount of formononetin = 283.95 ng;

Injection volume = 10 μl;

Amount of formononetin in sample = 28.395 ng/ μl = 28.395 μg/ ml

Therefore, since 8 oz of wet kudzu root was immersed in 16 oz ethanol to make a tincture, the tincture contained 28.395 μg/ ml of formononetin in solution.

Amount of isoflavones in Extract (μg/ ml) = [(amount of isoflavones obtained from HPLC in ng) / (injection volume in μl)

For meat samples:

Data obtained from HPLC of the meat sample is calculated in the following step- wise progression:

Puerarin = 237.98 ng ; Injection volume = 10 μl; thus

Amount of puerarin in the sample = 23.798 ng/ μl = 23.798 μg/ ml

2 gm of meat sample was diluted to 10 ml with DMSO to obtain 23.798 μg/ ml

1ml of the solution contained 23.798 μg of puerarin

10ml of the solution contained 237.98 μg of puerarin

2 gm of meat sample contained 237.98 μg/ ml of puerarin

Amount of Puerarin per gm of meat = 118.99 μg/ gm

Amount of isoflavones per gm of meat = [(amount of isoflavones from HPLC in ng / injection volume in μl) x 10ml dilution factor of meat] / 2 gm of meat sample

4.2.7 Basic hydrolysis of kudzu root extract Basic hydrolysis of kudzu root extract was performed according to the procedure of Delmonte and Rader (2006). Four milliliters of the extract was placed in a 5 ml

graduated glass stoppered volumetric flask and 300 μl of 2N NaOH was added. The

extract was swirled, and after 10 min 100 μl of glacial acetic acid was added. The

samples were diluted to a final volume of 5 ml with 50:50 H2O/MeCN solutions. The

solution was filtered through a 0.45μm PTFE membrane before HPLC analysis.

4.2.8 Acid hydrolysis of kudzu root extract Acid hydrolysis of kudzu root extract was similar to the procedure of Delmonte

and Rader (2006). Four milliliters of extract was placed in screw capped test tubes with

1ml of concentrated HCl. The tubes were heated for 2 h at 80°C in a water bath. The

tubes were chilled to room temperature (21°C), then 2 ml of the solution 50:5:45 NH4OH

(28–30% in H2O)/glacial acetic acid/DMSO was added. Because of the exothermic acid– base reaction associated with the preparation of this solution, it was carefully prepared under the hood by slow addition of reagents. The ratio between NH4OH and acetic acid

was adjusted in order to obtain a neutral pH (tested by pH paper) after addition to the hydrolyzed extract. The solution was filtered through a 0.45μm PTFE membrane before

HPLC analysis.

4.2.9 Preparation and cooking of beef patties with kudzu root extract Fresh lean beef was obtained from the Meat Science Laboratory at the

Department of Food Science Nutrition and Health Promotion, Mississippi State

University. A random sample from each replication was analyzed to determine fat

percentage using a near-infrared spectrometer (FoodScan Lab Analyzer Model 78800,

Foss Analytical, Eden Prairie, MN) that is AOAC (Association of Official Analytical

Chemists) approved Schilling and others (Schilling and others 2001). The fat and moisture content ranged between 7-8 % and 68-70%, respectively in fresh lean beef. The fresh lean beef was ground twice using a 1.27 cm plate in a Hobart meat grinder (80055

Mixer-grinder, Hollymatic Co., Countryside, IL). Three 2 pound batches of ground beef were randomly assigned to 0%, 1%, and 3% kudzu root extract treatments. For each treatment group, ground beef was hand mixed with 1% and 3% kudzu root crude extract which was received from Wild Pantry (Mother Nature Super Store, Tellico Plains, TN) and a control was made with no extract. The two treatment batches and control were then stuffed (Risco I-36016 Thiene, Vincenza, Italy) into 7.62 cm diameter plastic tubes

(Interstate Packaging, White Bluff, TN) and sealed at one end with metal clips to make logs of ground beef. The beef logs were labeled and frozen at -23°C until further evaluation. All equipment was cleaned between each treatment for all replications. The same process was repeated to make samples for second and third replicates and the gap between the replicates was 1 week.

4.2.9.1 Cooking of patties The frozen beef logs were removed from the plastic tubes and sliced into 1.27 cm thick patties (Butcher Boy ™ American Meat Equipment, LLC, Selmer, TN) with individual patties placed in a 3 mil plastic bag (Prime Source, 15.24 cm x 21.59 cm,

Kansas City, MO) and vacuum packaged (Model CV3HS, JVR Industries, Buffalo, NY) at 999 mbars. The frozen patties were thawed at room temperature for 15 min, weighed and then cooked on a griddle top stove (Griddle 442A, Toastmaster Inc., Booneville, 82

MO). The griddles were preheated to temperature of 176°C for 5 min prior to placement

of beef patties on the griddle. Each patty was cooked for 5 min on both sides, with an

additional 1 minute on each side until an internal temperature of 71°C was reached as

measured by a handheld digital thermometer (Model PT100 RTD, Omega Engineering

Inc, Bridgeport, NJ). After cooling at room temperature, the cooked patties were kept at -

23°C before further processing for HPLC analysis.

4.2.10 Extraction of isoflavones from uncooked and cooked beef patties The extraction of isoflavones from beef patties was performed as described by

Vranova (2005), who quantified soy isoflavones in meat products by HPLC. The

procedure was slightly modified to suit our objective eliminating the process of heating

(70°C) as was performed in Vranova’s protocol. Two beef patties were randomly selected

from either the uncooked or cooked treatment and control groups (0, 1% and 3% kudzu

root extract beef patties) and ground in a food processor (Cuisinart, Mini-Prep Plus, 3-

Cup Food Processor, New York, NY) to obtain a homogenous mixture. Two grams of the

homogenized sample was mixed with 20 ml of n-hexane in centrifuge tubes, thoroughly

vortex- mixed for 3 min and then sonicated at room temperature (21°C) for 30 min in a

water bath (Branson 1200 E4, Branson Co, Danbury, CT). The sample was centrifuged

for 10 min at 8500 rpm, and the hexane decanted leaving the meat sample. This procedure was repeated three times. The defatted meat sample was the mixed with 10 ml

of DMSO to dissolve the isoflavones. The mixture was sonicated for 30 min and

centrifuged at 8500 rpm. The isoflavones dissolved in DMSO solution were pipetted out

and filtered through 0.45μm PTFE membranes before HPLC analysis. Two samples per

treatment per replication were used for analysis. 83

4.3 Statistical analysis To assess the effect of cooking on isoflavone content in uncooked and cooked

beef patties formulated with kudzu root crude extract at 0%, 1% and 3%, the data was analyzed as a completely randomized design (CRD) with three replications and 6 treatments. Treatments included 1) Uncooked beef patty with 0% kudzu root extract; 2)

Cooked beef patty with 0% kudzu root extract; 3) Uncooked beef patty with 1% kudzu root extract; 4) Cooked beef patty with 1% kudzu root extract; 5) Uncooked beef patty with 3% kudzu root extract; 6) Cooked beef patty with 3% kudzu root extract. The data was analyzed using a one-way classification fixed effects model using PROC GLM with appropriate LSD multiple comparisons being obtained from LSMEANS (SAS 2002) and a P- value < 0.05 was considered as significant (Table 4.2).

Model: Yij = μ + τi + ε (i)j (4.1)

th where τi represents the effect of the i treatment, i = 1,…6; ε (i)j is the experimental error

2 associated with the source of variation due to replications, ε (i)j ~ N(0, σ ), i.i.d is assumed to be independent random variable, normally distributed with mean 0 and possesses variance component σ 2.

4.4 Results and discussion

4.4.1 Identification and quantification of isoflavones in kudzu root extract The chromatographic profiles of the standard solution containing reference isoflavones, and the kudzu root crude extract are given in Figure 4.1 A and B respectively. By comparison of RRT (relative retention time) values of authentic standards, 10 isoflavones were identified in the kudzu root crude extract. The isoflavones

identified were puerarin, diadzin, genistin, ononin, diadzein, glycitein, calycosin,

genistein, formononetin and biochanin A. Del Monte and Rader (2006) observed 22

isoflavones including the ten isoflavones identified in this study in a dietary supplement containing soy, red clover and kudzu. Sibao and others (2007) reported puerarin, diadzin, genistein, diadzein and formononetin in the root of kudzu plants cultivated in China.

Puerarin, diadzin, genistein and diadzein have been reported in kudzu root by other researchers as well (Zhang and others 2005; Prasain and others 2007; Chen and others

2001). In addition to the four isoflavones mentioned in the previous statement, Benlhabib

and others (2004) found biochanin A in kudzu root. Wu and others (2011) identified 24

isoflavones in kudzu root extract including those in this study through LC-UV(Liquid

Chromatography-Ultra Violet and processed MS/MS (Mass Spectrometry/ Mass

Spectrometry) methods. Wu and others’ study (2011) differed from this study in the use of a) finely ground powder from Pueraria lobata root b) differences in the extraction

process and c) use of UV/ MS (Ultra Violet/ Mass Spectrometry) method instead of

HPLC.

Figure 4.1 HPLC chromatogram of A) standard solution; B) kudzu root crude extract

A Standard solution with 10 reference isoflavones B 1 ml of kudzuroot extract diluted to 5 ml with DMSO

Figure 4.3 shows a HPLC chromatogram with 10 isoflavones in kudzu root

extract after acid and basic hydrolysis. Isoflavones in kudzu plant occur in various

chemical forms such as as aglycones (diadzein, genistein, glycitein, Biochanin A),

glycosides (puerarin, diadzin, genistin, glycitin) and malonate conjugates (glycoside

malonate of diadzein and genistein) (Benlhabib and others 2004; Wu and others 2011).

Daidzein is the aglycone form of puerarin and has been synthesized artiﬁcially (Chen and

others 2001). Often the isoflavones present in different forms in food material are

difficult to identify and quantify. Sometimes appropriate HPLC reference standards are

not commercially available, therefore isoflavones might volatilize in unidentified forms,

or compounds might not be chromatographically separated from other substances present

in the extract (Delmonte and Rader 2006). Acid and basic hydrolysis is used to break the

isoflavone derivatives into compounds that are easier to quantify. Figure 4.2 shows the

breakdown of isoflavone derivatives by acid and basic hydrolysis. Acid hydrolysis breaks

the bond between the isoflavones and the glucoside moieties, transforming all the

isoflavone derivatives into their aglycone forms. This procedure helps to quantify

isoflavones like prunetin, calycosin and prantension which are present in Red Clover in

glucoside form while only commercially available as aglycones (Klejdus and others

2001). Thus, acid hydrolysis converts the natural occurring glucosides into aglycones

which further assists in quantification. The basic hydrolysis breaks the ester bonds,

removing the acid group that are bonded to the sugar moiety of the isoflavones

glucosides, thus converting all isoflavone derivatives (e.g 6”-O-malonyl-β-glucoside) into their respective β-glucosides. Diadzein glucoside is diazin, glycitein glucoside is glycitin.

Thus isoflavone derivatives in the form of glucosides are easily quantified by basic

hydrolysis. In this study the 10 isoflavones found in the crude extract were also found after acid and basic hydrolysis further confirming the authenticity of the isoflavones.

There were few unidentified peaks seen in the crude extract after puerarin elution which were absent after acid hydrolysis (Figure 4.3). These peaks could be methoxy puerarin which might be digested after acid hydrolysis (detail analysis not performed in this study).

Figure 4.2 Scheme of acid and basic hydrolysis of isoflavone derivatives (Delmonte and Rader 2006)

Figure 4.3 HPLC chromatogram of A) kudzu root crude extract after acid hydrolysis; B) kudzu root crude extract after basic hydrolysis

Table 4.1 displays the amount of isoflavone content in kudzu root crude extract.

Puerarin was the most abundant (3.279 ± 0.26 mg/ml) isoflavone followed by diadzin

(407μg/ml). Puerarin and diadzein are mentioned as the two major isoflavones in kudzu

root. Kudzu root is also considered to be the main material source for extracting puerarin

for the medicinal industry in China (Sun and Sung 1998). The other 8 isoflavones ranged

between 51.96μg/ml (glycitein) to 7.215μg/ml (biochanin A). Prasain and others (2007)

obtained puerarin (14.5 mg/g) diadzein (0.27 mg/g), genistein (0.00) and diadzin (7.89

mg/g) and genistin (0.52 mg/gm) in an extract of kudzu root culture. Puerarin content was

five times more abundant in kudzu root culture (14.5 mg/g) than obtained (3.279 ± 0.26

mg/ml) in this study. Diadzein and genistein concentrations obtained in the present study

were more than obtained from kudzu root culture. The differences in the content of

isoflavones between these studies could be due to the differences in roots used, age of roots, and differences in the extraction / processing method. Seasonal variation in the isoflavones of kudzu root (Radix Pueraria) grown in China was studied to determine the optimum time to harvest the herb. The examination of 96 kudzu root samples of different age, harvested at different months clearly revealed that 3-yr old roots harvested in

January have the highest yield of isoflavonoid compounds (Sibao and others 2007). The optimum harvest time might differ for plants grown in the United States due to the differences in soil and climatic conditions. In this study the roots of kudzu plant were harvested in September from the area in Monroe County, TN, between Tellico Plains, TN

and Madisonville, TN. The exact age of kudzu root was not known, however the extraction center (Wild Pantry, Mother Nature’s Super Store, Tellico Plains, TN) confirmed the roots to be more than 2 yrs old.

Table 4.1 Isoflavone content (μg/ml dry weight) in (a) kudzu root crude extract; (b) kudzu root extract after acid hydrolysis; (c) kudzu root extract after basic hydrolysis

Isoflavones Crude extract ± SD Acid hydrolysis ±SD Basic hydrolysis ±SD (μg/ml) (n=4) (n=3) (n=3) Puerarin 3279.72± 261 1810.02 ± 160 2578.14 ± 101.3 Diadzin 23.8 ± 7.6 3.24 ± 0.04 35.09 ± 0.06 Genistin 10.09 ± 6.4 4.03 ± 0.43 8.018 ± 1.87 Ononin 10.97 ± 0.34 1.03 ± 0.04 8.35 ± 0.76 Diadzein 407.95 ± 204 297.35 ± 3.21 493.86 ± 98.99 Glycitein 51.96 ± 7.8 30.94 ± 3.24 42.43 ± 4.22 Calycosin 11.5 ± 0.25 8.23 ± 4.21 8.23 ± 2.33 Genistein 31.94 ± 10.8 2.98 ±0.03 28.45 ± 3.45 Formononetin 35.19 ± 12.49 32.03 ± 0.85 32.01 ± 0.04 Biochanin A 7.25 ± 5.82 2.13 ± 0.04 2.13 ± 0.05 SD, Standard deviation n=number of samples analyzed to calculate mean

Table 4.1 also shows the content of isoflavones in kudzu root extract after acid

and basic hydrolysis. The quantitation of puerarin, genistin, ononin, diadzein glycitein, calycosin and genistein in the unhydrolyzed crude extract or basic hydrolyzed extract appeared to be affected by the presence of co-eluting compounds that disappeared after acid hydrolysis. The findings are similar to others (Delmonte and Rader 2006; Wu and others 2011) who have reported decreased amount of total isoflavones after acid hydrolysis compared to unhydrolyzed crude extract. The quantity of diadzin detected after basic hydrolysis seemed to be numerically higher than that detected in the unhydrolyzed extract or following acid hydrolysis (23 versus 17 and 3.242 μg/ml) (Table

4.1). This might have been influenced by the presence of other non detected isoflavones

which converted into glucosides after basic hydrolysis.

4.4.2 Stability of Kudzu root extract isoflavones in beef patties during cooking Figure 4.4 and 4.5 show the HPLC chromatograms of uncooked and cooked beef patties with 0, 1 and 3% kudzu root crude extract. The qualitative analysis of the

chromatogram for uncooked and cooked control samples showed no elution of peaks,

confirming the absence of noise in the processing and identification process. Puerarin and

diadzein were the two major isoflavones recovered from treatment samples. The other

isoflavones detected were glycitein and genistein at a lower concentration. The other 6

isoflavones were at much lower concentrations in the kudzu root crude extract and

therefore could not be extracted from meat treatments at a detectable level.

Table 4.2 lists the amount of isoflavones recovered from uncooked and cooked

beef patties with 0, 1 and 3% crude root extract. The puerarin, diadzein and genistein

content in the 3% kudzu root extract beef patty was higher (P <0.05) than 1% kudzu root

extract beef patty. Cooking had no (P >0.05) affect on the isoflavones content as

compared to the uncooked beef patties. Results of this study are in agreement with other

researchers (Grun and others 2001; Hendrich and Murphy 2001b; Jackson and others

2002; Uzzan and Labuza 2004) who reported that isoflavones are not destroyed by heat

treatment during cooking, but rather losses are due to intra-conversions between the

different forms of isoflavones or leaching into the discarded cooking liquid. Other

researchers (Uzzan and Labuza 2004) observed qualitative and quantitative degradations

of isoflavones, if isoflavones were heated to higher temperatures (above 121°C) or longer

times than normal food processing conditions. Xu and others (2001) purified diadzin, 92

genistin and glycitin from toasted soy flour and heated samples up to 90 min at 95°C to

215°C and observed no significant change up to 110°C but temperature dependent degradation was found after 135°C. Genistein and diadzin was reported to decrease during storage of soy milk at 70°C to 90°C (Eisen and others 2003). Purified forms of diadzein and genistein incubated at 70 °C, 80 °C, 90 °C or autoclaved at 120°C for 20 min were reported to degrade at all temperatures (Uzzan and Labuza 2004). Overall the possibility of different degradation mechanisms are dependent on different heating conditions and environments, like high moisture -low temperature or low moisture and high temperature (Uzzan and Labuza 2004).

Table 4.2 Effect of cooking on isoflavone content (μg/gm, dry weight) in uncooked and cooked beef patties formulated with kudzu root crude extract at 0%, 1% and 3%.

Isoflavones (μg/gm) KRE 0% KRE 1% KRE 3% P value Uncooked Cooked Uncooked Cooked Uncooked Cooked Puerarin 0 c 0 c 45.6 b 44.1 b 141.2 a 129.1 a <0.0001 Diadzein 0 c 0 c 13.4 b 18.4b 37.1 a 33.9 a <0.0001 Glycitein 0 0 15.5 13.6 4.7 13.6 0.0762 Genistein 0 b 0 b 3.8 b 4.0 b 11.8 a 12.1 a 0.0058 a, b,c Means in the same row with different letters are significantly different (P< 0.05). KRE, Kudzu root extract

Figure 4.4 HPLC chromatograms of uncooked beef patties.

A) 0% kudzu root crude extract; B) 1% kudzu root crude extract; C) 3% kudzu root crude extract: (1) puerarin, (2) diadzein, (3) gylcitin, (4) genistein.

Figure 4.5 HPLC chromatograms of cooked beef patties.

A) 0% kudzu root crude extract; B) 1% kudzu root crude extract; C) 3% kudzu root crude extract: (1) puerarin, (2) diadzein, (3) gylcitin, (4) genistein.

4.5 Conclusions In this study, isoflavones present in kudzu root were identified and quantified.

Ten isoflavones, namely puerarin, diadzin, diadzein, genistin, genistein, glycitein, ononin, calycosin, biochaninA, and formononetin were identified in the extract. Puerarin and diadzein were the major isoflavones identified accounting for 95% of the isoflavones content. The concentration of puerarin and diadzein was approximately 3279.72 and

407.95 μg/ml, respectively, while other isoflavone concentrations ranged from approximately 7 to 51μg/ml in the tincture. Beef patties with 0, 1 and 3% kudzu root extract were assessed for isoflavone thermal stability during cooking. At concentrations of 1, and 3% of kudzu root extract in beef patties, four isoflavones, puerarin, diadzein, glycitein and genistein were detected in uncooked and cooked samples, with concentrations ranging from approximately 3.77 to 141.8μg/g of a beef pattie. Other non- identified isoflavones were considered to be diluted to undetectable levels in beef patties.

Isoflavone content of uncooked and cooked patties did not change (P >0.05) during cooking. Beef patties with 3% kudzu extract contained higher (P <0.05) isoflavone content than patties with 1% extract. The kudzu root isoflavone as a beef additive promises health benefits. Keung (2002) reported kudzu isoflavones to have antioxidative, anti-inflammatory and anti-cancerous properties. He also showed that isoflavones such as puerarin and diadzein suppress the desire for voluntary alcohol intake. The abundance of isoflavones in kudzu root qualifies it as a nutraceutical and demonstrates its potential for health benefits. However this is an area which needs to be further explored.

CHAPTER V

CONCLUSION

The kudzu (Pueraria lobata) plant is an edible leguminous vine. Even though the plant has been known to be widely used as a staple crop in Japan and China for centuries, there is limited literature to support its use in the United States’ main stream food products. This research focused on utilization of kudzu root starch and kudzu root extract in beef patties as a texturizer and a functional ingredient in beef patties.

Study I compared the effects of two types of starch (kudzu and potato) at three levels (2, 4, and 6%) in beef patties with respect to physical properties (pH, cooking yield, expressible moisture), chemical properties(protein, fat, moisture), texture profile analysis (hardness, springiness, cohesiveness, chewiness and gumminess), color analysis

(L*, a*, b*), scanning electron microscope images, and consumer acceptability

(appearance, aroma, flavor, texture and overall acceptability). A positive control was made with no starch and 5% water, and a negative control was made with no starch and no water. Averaged over starch levels, beef patties with potato starch were found to be higher (P<0.05) in moisture, lower (P<0.05) in fat and lower (P<0.05) in protein as compared to kudzu starch beef patties. Scanning electron microscope images indicated kudzu starch granules to be smaller in size than potato starch. Smaller granule size indicates that kudzu granules did not disturb the protein network as much as potato starch granules and thus improved the water binding properties of beef patties while maintaining 97

the compact nature of muscle fibers. Texture Profile Analysis revealed kudzu starch beef

patties to be significantly harder and gummier than potato starch patties. As expected,

increasing the starch levels in beef patties generally increased total moisture and

decreased expressible moisture, fat content, and protein content. K4, K6, P4 and P6

samples were significantly higher in cooking yield compared to NC and PC samples.

Certain beef patties with starches were lower in protein, lower in fat and higher in

moisture content compared to samples with no starch and no water. Uncooked beef

patties with starch were significantly lighter (whiter) in color than PC and NC samples.

Consumer acceptability tests showed no significant differences among treatment or

control samples for appearance, aroma, flavor, and texture liking, however, averaged over

starch levels, potato starch (5.7) scored higher (P <0.05) overall liking scores compared

to kudzu (5.3) starch patties. The overall liking score of all samples ranged between 5

“neither like nor dislike” and 6 “like slightly”. However, a difference of “0.3” in value on

a 9 point hedonic scale (1=dislike extremely; 9= like extremely) for the overall liking

score between potato and kudzu samples at significance level P= 0.0431 is of relatively

little practical importance.

In study II, isoflavones present in kudzu root were identified and quantified.

Kudzu root extract was prepared by ethanol extraction at room temperature using kudzu

roots harvested from the area in Monroe County, TN, between Tellico Plains, TN and

Madisonville, TN. Ten isoflavones, namely puerarin, diadzin, diadzein, genistin,

genistein, glycitein, ononin, calycosin, biochaninA, and formononetin were identified in the kudzu root extract. Puerarin and diadzein were the major isoflavones identified accounting for 95% of the isoflavone content. The concentration of puerarin and diadzein

was approximately 3279.72 and 407.95 μg/ml, respectively, while other isoflavone

concentrations ranged from approximately 7 to 51μg/ml in the tincture. Beef patties with

0, 1 and 3% kudzu root extract were assessed for isoflavone stability during cooking. At

concentrations of 1, and 3% kudzu root extract in beef patties, four isoflavones, puerarin,

diadzein, glycitein and genistein were detected in uncooked and cooked samples with

concentrations ranging from approximately 3.77 to 141.8μg/g of beef pattie. Other non-

identified isoflavones were concluded to be diluted to undetectable levels in beef pattie

samples. Isoflavone content of uncooked and cooked patties did not change significantly

during cooking. As expected, beef patties with 3% kudzu extract contained higher

(P<0.05) isoflavone content than patties with 1% extract. The kudzu root isoflavone as a

beef additive shows potential health benefits. Kudzu isoflavones are reported to have

antioxidative, anti-inflammatory and anti-cancerous properties. Puerarin and diadzein are

reported to suppress the desire for voluntary alcohol intake (Kueng, 2002). Puerarin is

reported to provide a therapeutic effect against non-alcoholic fatty liver in rats and

preventive against alcohol induced osteonecrosis (bone death) in mice (Kueng, 2002).

The abundance of isoflavones in kudzu root with several medicinal properties qualifies it

as a nutraceutical. However this is an area which needs to be explored further.

This research demonstrated that the addition of kudzu and potato starch in beef

patties showed similar responses for most sensory and physicochemical properties,

therefore kudzu starch shows the potential for use as an alternative starch in food products; however more research is needed to confirm acceptable performance. Most of the kudzu powder sold in the United States is imported from Japan. The production and processing of kudzu starch in Japan takes about 60-90 days which increases the final cost.

An average cost of potato starch ranges between 16.5 cents to 3.3 dollars per pound whereas kudzu starch costs between 35.30 – 42.65 dollars per pound. The price of starch being one of the precluding factors for kudzu use, an advance processing facility coupled with optimized logistics with abundant kudzu availability in the United States would be

highly favorable to increase the yield and reduce cost of production. This study

substantiates kudzu starch potentialities for future investigations in areas like- fat

replacers or texturizers, optimization of starch level with water, using modified forms of

starch (fat composites, prior-acid or base treated) and application suitability as an

economic advantage leading to optimum manufacturing costs. Kudzu root extract’s

richness in isoflavone content and isoflavone stability during cooking warrants further

studies with sensory/consumer testing and as an ingredient in other applications(yogurt,

snacks, ready to eat frozen products) providing health/nutritional benefits and convening

novel space in nutraceutical markets. The information of this research will aid other food

professionals in new product development using kudzu. Kudzu as a resource is abundant

and complete eradication in the near future is not possible due to the cost of control and

consistent propagating growth of the plant. Other research professionals are also in search

of kudzu usage as biofuels, pharmacological purposes and agricultural or food sources.

Therefore, the kudzu plant as an economic commodity warrants further research to

hopefully maximize its economic potential as a food and/or health benefits.

100

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APPENDIX A

SAS OUTPUTS

109

A.1 SAS outputs for Table 3.2, Table 3.4, Table 3.7, Table 3.9 and Table 3.11

pH Tests of Hypotheses Using the Type III MS for Rep(starcht*starchc) as an Error Term

Source DF Type III SS Mean Square F Value Pr > F

starchtype 1 0.00006944 0.00006944 0.03 0.8578 starchconc 2 0.76550556 0.38275278 184.71 <.0001 starchtyp*starchconc 2 0.00010556 0.00005278 0.03 0.9749

T Comparison Lines for Least Squares Means of starchconc LS-means with the same letter are not significantly different. LSMEAN 110 pH LSMEAN starchconc Number A 5.7250000 4 2 A A 5.6908333 2 1

B 5.4000000 6 3

COOKING YIELD Dependent Variable: Cookingyield

Tests of Hypotheses Using the Type III MS for Rep(starcht*starchc) as an Error Term

Source DF Type III SS Mean Square F Value Pr > F

starchtype 1 16.7304618 16.7304618 0.31 0.5953 starchconc 2 63.4221988 31.7110994 0.60 0.5807 starchtyp*starchconc 2 173.6930141 86.8465071 1.63 0.2717

Expressible moisture

Dependent Variable: Expresssiblemoisture

Tests of Hypotheses Using the Type III MS for Rep(starcht*starchc) as an Error Term

Source DF Type III SS Mean Square F Value Pr > F

111 starchtype 1 0.96581614 0.96581614 6.56 0.0249 starchconc 2 1.35792247 0.67896123 4.61 0.0326 starchtyp*starchconc 2 0.18464989 0.09232495 0.63 0.5505

T Comparison Lines for Least Squares Means of starchtype

LS-means with the same letter are not significantly different.

Expresssiblemoisture LSMEAN LSMEAN starchtype Number

A 1.2788189 Kudzu 1

B 1.0450454 potat 2

LS-means with the same letter are not significantly different.

Expresssiblemoisture LSMEAN LSMEAN starchconc Number

A 1.3029356 2 1 A B A 1.2093758 4 2 B B 0.9734850 6 3

Chemical analysis

Fat

112 Dependent Variable: fat

Tests of Hypotheses Using the Type III MS for Rep(starcht*starchc) as an Error Term

Source DF Type III SS Mean Square F Value Pr > F

starchtype 1 5.68890032 5.68890032 17.95 0.0012 starchconc 2 34.49589933 17.24794967 54.41 <.0001 starchtyp*starchconc 2 1.69737933 0.84868967 2.68 0.1093

T Comparison Lines for Least Squares Means of starchtype

LS-means with the same letter are not significantly different.

LSMEAN fat LSMEAN starchtype Number

A 9.0657407 Kud 1

B 8.2855556 pot 2

T Comparison Lines for Least Squares Means of starchconc

LS-means with the same letter are not significantly different.

LSMEAN fat LSMEAN starchconc Number

113 A 9.7300000 2 1

B 8.8633333 4 2

C 7.4336111 6 3

Protein

Dependent Variable: pro

Tests of Hypotheses Using the Type III MS for Rep(starcht*starchc) as an Error Term

Source DF Type III SS Mean Square F Value Pr > F

starchtype 1 23.5675080 23.5675080 50.47 <.0001 starchconc 2 117.4061507 58.7030753 125.71 <.0001

starchtyp*starchconc 2 1.0525893 0.5262947 1.13 0.3560

T Comparison Lines for Least Squares Means of starchtype

LS-means with the same letter are not significantly different.

pro LSMEAN LSMEAN starchtype Number

A 26.30019 Kud 1

B 24.71222 pot 2

The GLM Procedure Least Squares Means for effect starchconc 114 Pr > |t| for H0: LSMean(i)=LSMean(j)

T Comparison Lines for Least Squares Means of starchconc

LS-means with the same letter are not significantly different.

pro LSMEAN LSMEAN starchconc Number

A 27.69417 2 1

B 25.42917 4 2

C 23.39528 6 3

Moisture Dependent Variable: mois

Tests of Hypotheses Using the Type III MS for Rep(starcht*starchc) as an Error Term

Source DF Type III SS Mean Square F Value Pr > F

starchtype 1 27.34092821 27.34092821 23.87 0.0004 starchconc 2 27.85378433 13.92689217 12.16 0.0013 starchtyp*starchconc 2 3.66142433 1.83071217 1.60 0.2424

T Comparison Lines for Least Squares Means of starchtype

LS-means with the same letter are not significantly different.

115 mois LSMEAN LSMEAN starchtype Number

A 64.36222 pot 2

B 62.65185 Kud 1

T Comparison Lines for Least Squares Means of starchconc

LS-means with the same letter are not significantly different.

mois LSMEAN LSMEAN starchconc Number

A 64.62194 6 3

B 63.34167 4 2 B B 62.55750 2 1 Color analysis l: Tests of Hypotheses Using the Type III MS for Rep(starcht*starchc) as an Error Term

Source DF Type III SS Mean Square F Value Pr > F

starchtype 1 459.1123358 459.1123358 56.36 <.0001 starchconc 2 131.2449049 65.6224525 8.06 0.0061 starchtyp*starchconc 2 68.4051494 34.2025747 4.20 0.0415

a: Tests of Hypotheses Using the Type III MS for Rep(starcht*starchc) as an Error Term 116

Source DF Type III SS Mean Square F Value Pr > F

starchtype 1 0.73339506 0.73339506 0.03 0.8677 starchconc 2 20.50616420 10.25308210 0.40 0.6760 starchtyp*starchconc 2 1.40550494 0.70275247 0.03 0.9727

b: The GLM Procedure Dependent Variable: b

Tests of Hypotheses Using the Type III MS for Rep(starcht*starchc) as an Error Term

Source DF Type III SS Mean Square F Value Pr > F

starchtype 1 121.8013636 121.8013636 4.58 0.0537 starchconc 2 33.2814309 16.6407154 0.63 0.5517 starchtyp*starchconc 2 18.7239642 9.3619821 0.35 0.7105

TEXTURE PROFILE ANALYSIS

117 Dependent Variable: Hard

Tests of Hypotheses Using the Type III MS for Rep(starcht*starchc) as an Error Term

Source DF Type III SS Mean Square F Value Pr > F

starchtype 1 109.7174222 109.7174222 5.32 0.0397 starchconc 2 7.8271444 3.9135722 0.19 0.8295 starchtyp*starchconc 2 0.9428778 0.4714389 0.02 0.9774

The GLM Procedure

T Comparison Lines for Least Squares Means of starchtype

LS-means with the same letter are not significantly different.

Hard LSMEAN

LSMEAN starchtype Number

A 9.3416667 Kud 1

B 6.8727778 Pot 2

The GLM Procedure

Dependent Variable: cohes

Tests of Hypotheses Using the Type III MS for Rep(starcht*starchc) as an Error Term

Source DF Type III SS Mean Square F Value Pr > F

118 starchtype 1 0.28934010 0.28934010 6.98 0.0268 starchconc 2 0.14185600 0.07092800 1.71 0.2344 starchtyp*starchconc 2 0.22755287 0.11377643 2.75 0.1172

T Comparison Lines for Least Squares Means of starchtype

LS-means with the same letter are not significantly different.

cohes LSMEAN LSMEAN starchtype Number

A 0.43486111 Kud 1

B 0.29311111 Pot 2

Dependent Variable: spring Tests of Hypotheses Using the Type III MS for Rep(starcht*starchc) as an Error Term

Source DF Type III SS Mean Square F Value Pr > F

starchtype 1 0.31494348 0.31494348 1.84 0.2085 starchconc 2 0.54467250 0.27233625 1.59 0.2567 starchtyp*starchconc 2 0.94364270 0.47182135 2.75 0.1169

Dependent Variable: Gumm Tests of Hypotheses Using the Type III MS for Rep(starcht*starchc) as an Error Term

Source DF Type III SS Mean Square F Value Pr > F

119 starchtype 1 46.51211111 46.51211111 10.55 0.0100 starchconc 2 6.88839976 3.44419988 0.78 0.4867 starchtyp*starchconc 2 6.53965847 3.26982924 0.74 0.5034

The GLM Procedure T Comparison Lines for Least Squares Means of starchtype

LS-means with the same letter are not significantly different.

Gumm LSMEAN LSMEAN starchtype Number

A 3.7035556 Kud 1

B 1.9063333 Pot 2

Dependent Variable: Chewi

Tests of Hypotheses Using the Type III MS for Rep(starcht*starchc) as an Error Term

Source DF Type III SS Mean Square F Value Pr > F

starchtype 1 50.49039893 50.49039893 41.73 0.0001 starchconc 2 22.91141482 11.45570741 9.47 0.0061 starchtyp*starchconc 2 19.42677933 9.71338966 8.03 0.0100

120 SENSORY ANALYSIS Dependent Variable: App

Tests of Hypotheses Using the Type III MS for Rep(starcht*starchc) as an Error Term

Source DF Type III SS Mean Square F Value Pr > F

starchtype 1 10.97652567 10.97652567 1.97 0.1860 starchconc 2 9.53137260 4.76568630 0.85 0.4498 starchtyp*starchconc 2 6.34826767 3.17413383 0.57 0.5806

Dependent Variable: Aro

Tests of Hypotheses Using the Type III MS for Rep(starcht*starchc) as an Error Term

Source DF Type III SS Mean Square F Value Pr > F

starchtype 1 2.86075372 2.86075372 0.44 0.5186 starchconc 2 9.60076775 4.80038388 0.74 0.4967 starchtyp*starchconc 2 1.28578206 0.64289103 0.10 0.9061

Dependent Variable: Tex

Tests of Hypotheses Using the Type III MS for Rep(starcht*starchc) as an Error Term

Source DF Type III SS Mean Square F Value Pr > F

121 starchtype 1 14.68979757 14.68979757 2.47 0.1420 starchconc 2 9.31197638 4.65598819 0.78 0.4791 starchtyp*starchconc 2 3.73879221 1.86939611 0.31 0.7361

Dependent Variable: Flv

Tests of Hypotheses Using the Type III MS for Rep(starcht*starchc) as an Error Term

Source DF Type III SS Mean Square F Value Pr > F

starchtype 1 14.63263460 14.63263460 3.34 0.0927 starchconc 2 3.17279183 1.58639591 0.36 0.7038 starchtyp*starchconc 2 8.11768180 4.05884090 0.93 0.4229 Dependent Variable: OA

Tests of Hypotheses Using the Type III MS for Rep(starcht*starchc) as an Error Term

Source DF Type III SS Mean Square F Value Pr > F

starchtype 1 17.01870532 17.01870532 5.11 0.0431 starchconc 2 0.65429700 0.32714850 0.10 0.9071 starchtyp*starchconc 2 3.19098180 1.59549090 0.48 0.6306

T Comparison Lines for Least Squares Means of starchtype

LS-means with the same letter are not significantly different.

122 LSMEAN OA LSMEAN starchtype Number

A 5.7121088 pot 2

B 5.3862736 kud 1

A.2 SAS outputs for Table 3.3, Table 3.8, Table 3.10 and Table 3.12 Dependent Variable: pH

Tests of Hypotheses Using the Type III MS for Rep(Treatment) as an Error Term Source DF Type III SS Mean Square F Value Pr > F Treatment 7 0.93369167 0.13338452 7.19 0.0006

Dependent Variable: Cookingyield

Tests of Hypotheses Using the Type III MS for Rep(Treatment) as an Error Term Source DF Type III SS Mean Square F Value Pr > F Treatment 7 3005.714688 429.387813 9.47 0.0001

Dependent Variable: Expresssiblemoisture

Tests of Hypotheses Using the Type III MS for Rep(Treatment) as an Error Term Source DF Type III SS Mean Square F Value Pr > F Treatment 7 79.67941560 11.38277366 2.72 0.0460

Dependent Variable: pro

Tests of Hypotheses Using the Type III MS for Rep(Treatment) as an Error Term 123

Source DF Type III SS Mean Square F Value Pr > F Treatment 15 1415.697950 94.379863 188.94 <.0001

Dependent Variable: fat

Tests of Hypotheses Using the Type III MS for Rep(Treatment) as an Error Term Source DF Type III SS Mean Square F Value Pr > F Treatment 15 95.22519621 6.34834641 12.48 <.0001

Dependent Variable: mois

Tests of Hypotheses Using the Type III MS for Rep(Treatment) as an Error Term Source DF Type III SS Mean Square F Value Pr > F Treatment 15 808.5069502 53.9004633 47.68 <.0001

Dependent Variable: Hard

Tests of Hypotheses Using the Type III MS for Rep(Treatment) as an Error Term Source DF Type III SS Mean Square F Value Pr > F Treatment 7 120.0836292 17.1548042 0.89 0.5352

Dependent Variable: Chewi

Tests of Hypotheses Using the Type III MS for Rep(Treatment) as an Error Term Source DF Type III SS Mean Square F Value Pr > F Treatment 7 43.54811530 6.22115933 16.65 <.0001

Dependent Variable: spring

124 Tests of Hypotheses Using the Type III MS for Rep(Treatment) as an Error Term Source DF Type III SS Mean Square F Value Pr > F Treatment 7 1.00317988 0.14331141 3.25 0.0318

Dependent Variable: Gumm

Tests of Hypotheses Using the Type III MS for Rep(Treatment) as an Error Term Source DF Type III SS Mean Square F Value Pr > F Treatment 7 81.22347837 11.60335405 3.44 0.0262

Dependent Variable: cohes

Tests of Hypotheses Using the Type III MS for Rep(Treatment) as an Error Term Source DF Type III SS Mean Square F Value Pr > F Treatment 7 0.86712830 0.12387547 3.63 0.0215

Dependent Variable: App

Tests of Hypotheses Using the Type III MS for Rep(Treatment) as an Error Term Source DF Type III SS Mean Square F Value Pr > F Treatment 6 31.87106732 5.31184455 1.06 0.4292

Dependent Variable: Aro

Tests of Hypotheses Using the Type III MS for Rep(Treatment) as an Error Term Source DF Type III SS Mean Square F Value Pr > F Treatment 6 16.84131037 2.80688506 0.48 0.8114

Dependent Variable: Tex

Tests of Hypotheses Using the Type III MS for Rep(Treatment) as an Error Term 125 Source DF Type III SS Mean Square F Value Pr > F Treatment 6 73.80575297 12.30095883 2.40 0.0828

Dependent Variable: Flv

Tests of Hypotheses Using the Type III MS for Rep(Treatment) as an Error Term Source DF Type III SS Mean Square F Value Pr > F Treatment 6 42.97733163 7.16288861 1.64 0.2080

Dependent Variable: OA

Tests of Hypotheses Using the Type III MS for Rep(Treatment) as an Error Term Source DF Type III SS Mean Square F Value Pr > F Treatment 6 37.64091253 6.27348542 1.63 0.2111

Dependent Variable: l

Source DF Type III SS Mean Square F Value Pr > F Treatment 7 134.0516579 19.1502368 37.06 <.0001 Rep 2 4.9703316 2.4851658 4.81 0.0257

Dependent Variable: a Source DF Type III SS Mean Square F Value Pr > F Treatment 7 3.93861888 0.56265984 0.51 0.8102 Rep 2 37.74855194 18.87427597 17.21 0.0002 Dependent Variable: b Source DF Type III SS Mean Square F Value Pr > F Treatment 7 28.26250605 4.03750086 8.43 0.0004 Rep 2 44.88756514 22.44378257 46.83 <.0001

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APPENDIX B

APPROVED INSTITUTIONAL REVIEW BOARD (IRB) FORM

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APPENDIX C

INFORMED CONSENT FORM

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APPENDIX D

SCORE SHEET FOR CONSUMER ACCEPTABILITY TEST

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CONSUMER ACCEPTANCE TEST

Samples: Beef Patties Date:______

Please taste each beef patty sample in the order listed. After chewing if you do not wish to swallow the sample, you may expectorate it in the cup and rinse with the water provided. Rate each sample in each of the five categories listed. Each column will need one check mark if you choose to evaluate all samples.

212 320 411 511 633 753 898 Appearance Like extremely Like very much Like moderately Like slightly Neither like or dislike Dislike slightly Dislike moderately Dislike very much Dislike extremely

212 320 411 511 633 753 898 Aroma Like extremely Like very much Like moderately Like slightly Neither like or dislike Dislike slightly Dislike moderately Dislike very much Dislike extremely

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212 320 411 511 633 753 898 Texture Like extremely Like very much Like moderately Like slightly Neither like or dislike Dislike slightly Dislike moderately Dislike very much Dislike extremely

212 320 411 511 633 753 898 Overall Flavor Like extremely Like very much Like moderately Like slightly Neither like or dislike Dislike slightly Dislike moderately Dislike very much Dislike extremely

212 320 411 511 633 753 898 Overall Acceptability Like extremely Like very much Like moderately Like slightly Neither like or dislike Dislike slightly Dislike moderately Dislike very much Dislike extremely

Any comment

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