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Detoxification, Nutritive Value, and Anthelmintic Properties of Mucuna pruriens

Permanent Link: http://ufdc.ufl.edu/UFE0022472/00001

Material Information

Title: Detoxification, Nutritive Value, and Anthelmintic Properties of Mucuna pruriens
Physical Description: 1 online resource (132 p.)
Language: english
Creator: Huisden, Christiaan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: anthelmintic, bean, detoxification, dopamine, feed, food, haemonchus, huisden, ldopa, levodopa, monogastric, mucuna, nutraceutical, nutritional, nutritive, parkinson, rat, safety, seed, sheep, silage, solvent, sonication, toxic, velvet
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The Mucuna pruriens bean has high protein and starch contents, but also contains 3, 4 -dihydroxy-L-phenylalanine (L-Dopa), which has pharmaceutical properties, but is toxic when ingested by monogastrics. Experiments were conducted to detoxify Mucuna for monogastrics, and to evaluate its anthelmintic effect in ruminants. Experiment 1a) examined how long it takes to decrease the pH of ensiled Mucuna to < 4.6, the typical minimum pH for ensiled legumes. Crushed beans (6 mm) were ensiled for 0, 3, 7, 21, and 28 days. A pH of 4.5 and an L-Dopa concentration of 1.3% (54% reduction) were recorded after 28 days of ensiling. Experiment 1b) determined the effect of particle size (2, 4, or 6 mm) of ensiled Mucuna on L-Dopa concentration and on fermentation and nutritional characteristics. Ensiling 1- or 6-mm particles reduced the L-Dopa concentration by about 60% while preserving most nutrients. Experiment 2 studied the effects of extraction in solutions of acetic acid (ACD, pH 3) or sodium hydroxide (ALK, pH 11) for 8 hours or sonication (SON) for 5 minutes on the L-Dopa concentration and nutritional composition of finely (1 mm) or coarsely (6 mm) ground Mucuna beans. All extraction methods reduced the L-Dopa concentration of fine particles to safe levels ( < 0.4%) but increased their neutral detergent fiber (NDF) and starch concentrations and decreased their water-soluble carbohydrate (WSC) and crude protein (CP) concentrations. Extraction methods were less effective at reducing the L-Dopa in coarse particles and had inconsisent effects on their nutritional composition. Experiment 3 evaluated the effect of feeding detoxified Mucuna beans on the performance, behavior, and health of 60 Sprague-Dawley rats randomly assigned to five treatments. Dietary treatments consisted of a commercial rat chow (CON) or diets in which 10% of the rat chow was replaced with either undetoxified Mucuna (MUC), or Mucuna detoxified by ACD or ALK extraction, or ensiling for 28 days (SIL). Compared to CON, Mucuna-based diets gave similar feed intake and weight gain. No behavioral abnormalities were caused by any of the diets in open field analyses on days 3 and 10 but when data for both days was collectively analyzed, all Mucuna-based diets exhibited less locomotion than control rats. The decrease in activity was numerically less in rats fed ACD and SIL diets than in those fed MUC and ALK. Feeding MUC caused splenomegaly and monocytosis, and reduced blood phosphorus concentrations relative to CON, but detoxification of Mucuna prevented these effects. Experiment 4 determined if ingestion of Mucuna beans reduces helminth parasite infestation in lambs. Thirty-six Dorper x Katahdin ram lambs (28.8 + 5 kg body weight) were randomly allocated to three treatments: a cottonseed meal control diet, a diet in which Mucuna replaced cottonseed meal, and a treatment that involved administering levamisole (2 ml/45.4 kg) to lambs fed the control diet. Diets were formulated to be isonitrogenous (14% CP) and isocaloric (64% total digestible nutrients). Lambs were challenged 3 times per week for 2 weeks by gavage with infectious H. contortus larvae. Unlike levamisole treatment, Mucuna intake did not affect (P < 0.05) fecal egg counts (412 vs. 445 eggs/g) or abomasal worm counts (958 vs. 1170 total worms), though a numerical (P > 0.10) reduction was evident. Neither levamisole nor Mucuna treatment affected anemia indicators, daily feed intake, weight gain or dressing. In conclusion, Mucuna intake did not reduce helminth parasite infection in lambs.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Christiaan Huisden.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Adesogan, Adegbola T.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022472:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022472/00001

Material Information

Title: Detoxification, Nutritive Value, and Anthelmintic Properties of Mucuna pruriens
Physical Description: 1 online resource (132 p.)
Language: english
Creator: Huisden, Christiaan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: anthelmintic, bean, detoxification, dopamine, feed, food, haemonchus, huisden, ldopa, levodopa, monogastric, mucuna, nutraceutical, nutritional, nutritive, parkinson, rat, safety, seed, sheep, silage, solvent, sonication, toxic, velvet
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The Mucuna pruriens bean has high protein and starch contents, but also contains 3, 4 -dihydroxy-L-phenylalanine (L-Dopa), which has pharmaceutical properties, but is toxic when ingested by monogastrics. Experiments were conducted to detoxify Mucuna for monogastrics, and to evaluate its anthelmintic effect in ruminants. Experiment 1a) examined how long it takes to decrease the pH of ensiled Mucuna to < 4.6, the typical minimum pH for ensiled legumes. Crushed beans (6 mm) were ensiled for 0, 3, 7, 21, and 28 days. A pH of 4.5 and an L-Dopa concentration of 1.3% (54% reduction) were recorded after 28 days of ensiling. Experiment 1b) determined the effect of particle size (2, 4, or 6 mm) of ensiled Mucuna on L-Dopa concentration and on fermentation and nutritional characteristics. Ensiling 1- or 6-mm particles reduced the L-Dopa concentration by about 60% while preserving most nutrients. Experiment 2 studied the effects of extraction in solutions of acetic acid (ACD, pH 3) or sodium hydroxide (ALK, pH 11) for 8 hours or sonication (SON) for 5 minutes on the L-Dopa concentration and nutritional composition of finely (1 mm) or coarsely (6 mm) ground Mucuna beans. All extraction methods reduced the L-Dopa concentration of fine particles to safe levels ( < 0.4%) but increased their neutral detergent fiber (NDF) and starch concentrations and decreased their water-soluble carbohydrate (WSC) and crude protein (CP) concentrations. Extraction methods were less effective at reducing the L-Dopa in coarse particles and had inconsisent effects on their nutritional composition. Experiment 3 evaluated the effect of feeding detoxified Mucuna beans on the performance, behavior, and health of 60 Sprague-Dawley rats randomly assigned to five treatments. Dietary treatments consisted of a commercial rat chow (CON) or diets in which 10% of the rat chow was replaced with either undetoxified Mucuna (MUC), or Mucuna detoxified by ACD or ALK extraction, or ensiling for 28 days (SIL). Compared to CON, Mucuna-based diets gave similar feed intake and weight gain. No behavioral abnormalities were caused by any of the diets in open field analyses on days 3 and 10 but when data for both days was collectively analyzed, all Mucuna-based diets exhibited less locomotion than control rats. The decrease in activity was numerically less in rats fed ACD and SIL diets than in those fed MUC and ALK. Feeding MUC caused splenomegaly and monocytosis, and reduced blood phosphorus concentrations relative to CON, but detoxification of Mucuna prevented these effects. Experiment 4 determined if ingestion of Mucuna beans reduces helminth parasite infestation in lambs. Thirty-six Dorper x Katahdin ram lambs (28.8 + 5 kg body weight) were randomly allocated to three treatments: a cottonseed meal control diet, a diet in which Mucuna replaced cottonseed meal, and a treatment that involved administering levamisole (2 ml/45.4 kg) to lambs fed the control diet. Diets were formulated to be isonitrogenous (14% CP) and isocaloric (64% total digestible nutrients). Lambs were challenged 3 times per week for 2 weeks by gavage with infectious H. contortus larvae. Unlike levamisole treatment, Mucuna intake did not affect (P < 0.05) fecal egg counts (412 vs. 445 eggs/g) or abomasal worm counts (958 vs. 1170 total worms), though a numerical (P > 0.10) reduction was evident. Neither levamisole nor Mucuna treatment affected anemia indicators, daily feed intake, weight gain or dressing. In conclusion, Mucuna intake did not reduce helminth parasite infection in lambs.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Christiaan Huisden.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Adesogan, Adegbola T.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022472:00001


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DETOXIFICATION, NUTRITIVE VALUE, A ND ANTHELMINTIC PROPERTIES OF Mucuna pruriens By CHRISTIAAN MAX HUISDEN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1

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2008 Christiaan Max Huisden 2

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To my family: The love of my life, my soul mate, and wife Andrea Feaster Huisden, and our four wonderful sons, Raoul Max Franklin Huisden (12), Christiaan Henry Huisden (5), John Franklin Sjaak Huisden (2), and Carlo Dennis Huisden (1). A sp ecial word of thanks to my precious wife, Andrea; without her unfailing love, patience, wisdom, support, and encouragement this accomplishment would not exist. 3

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ACKNOWLEDGMENTS The author wishes to proclaim that this work was made possible only by the grace, love, and guidance of almighty God. Without Him I am nothing, yet through Jesus Christ all things are possible. I hereby express enormous gratitude to my committee chai r, Dr. Adegbola T. Adesogan, and members Dr. Nancy J. Szabo, Dr Veronika D. Butterweck, and Dr. Lokenga Badinga, for their excellent guidance throughout my Ph.D program. I greatly appreciate the assistance of all my laboratory colleagues and es pecially the people who were directly involved in the completion of the research requirements fo r this degree: Dr. Jack M. Gaskin, Dr. Ademola M. Raji, Dr. Charles H. Courtney III, Dr. Lv Yongning, Elizabeth Maxwell, Rolando Schlaefli, Taewon Kang, Brittany Grube, Diane Heaton-Jones, Raoul M.F. Huisden, and Dr. E. Greiner and his laboratory staff. 4

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES.........................................................................................................................9 ABSTRACT...................................................................................................................................10 CHAPTER 1 INTRODUCTION................................................................................................................. .12 2 LITERATURE REVIEW.......................................................................................................14 Introduction................................................................................................................... ..........14 Taxonomy and Characteristics of Mucuna pruriens ..............................................................15 Classification...................................................................................................................15 Species Description.........................................................................................................16 Current Uses of Mucuna pruriens ..........................................................................................17 Nutraceutical Versatility..................................................................................................17 Antioxidant property................................................................................................18 Antivenin property...................................................................................................19 Fertility-enhancing property.....................................................................................19 Growth-promoting property.....................................................................................20 Anthelmintic property..............................................................................................20 Mucuna pruriens as a Food and Feed Source.................................................................21 Nutritional Value.............................................................................................................. ......23 Protein Concentration......................................................................................................23 Other Nutrients................................................................................................................26 Comparison to Other Legumes........................................................................................27 Antinutritional and Toxic Properties......................................................................................29 Tannins............................................................................................................................31 Proanthocyanidins....................................................................................................32 Hydrolyzable tannins................................................................................................32 L-Dopa.............................................................................................................................33 Pharmacodynamics of L-Dopa in humans...............................................................34 Adverse effects of L-Dopa on humans.....................................................................36 Adverse effects of L-Dopa on monogastric livestock..............................................37 Safe levels of dietary Mucuna L-Dopa...................................................................................39 Detoxification of Mucuna pruriens ........................................................................................41 Boiling.............................................................................................................................42 Roasting...........................................................................................................................43 Ensiling....................................................................................................................... .....44 Solvent Extraction...........................................................................................................44 5

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Solubility in water....................................................................................................45 Solubility in alkaline and acid solvents....................................................................47 Sonication........................................................................................................................48 Statement of Objectives........................................................................................................ ..48 3 EFFECT OF ENSILING ON L-DOPA AND NUTRITIONAL VALUE OF Mucuna pruriens ...................................................................................................................................50 Introduction................................................................................................................... ..........50 Materials and Methods...........................................................................................................52 Effects of Ensiling Duration............................................................................................53 Effects of Particle Size of Ensiled Mucuna .....................................................................54 Chemical Analysis...........................................................................................................54 Statistical Analysis..........................................................................................................5 6 Results.....................................................................................................................................57 Effects of Ensiling Duration............................................................................................57 Effects of Particle Size of Ensiled Mucuna .....................................................................58 Discussion...............................................................................................................................60 Conclusion..............................................................................................................................63 4 EFFECT OF SONICATION AND SO LVENT EXTRACTION ON L-DOPA AND NUTRITIONAL VALUE OF Mucuna pruriens ....................................................................65 Introduction................................................................................................................... ..........65 Materials and Methods...........................................................................................................67 Extraction Methods.........................................................................................................67 Statistical Analysis..........................................................................................................6 9 Results.....................................................................................................................................69 Discussion...............................................................................................................................71 Conclusion..............................................................................................................................75 5 BEHAVIORAL, PERFORMANCE, AND PHYSIOLOGICAL RESPONSES OF RATS FED DETOXIFIED Mucuna pruriens ........................................................................77 Introduction................................................................................................................... ..........77 Materials and Methods...........................................................................................................79 Mucuna Detoxification....................................................................................................79 Detoxification through acid or alkali solvent extraction..........................................79 Detoxification through ensiling................................................................................80 Analysis of L-Dopa.........................................................................................................81 Nutritional Value Analysis..............................................................................................81 Dietary Treatments..........................................................................................................82 Animals and Measurements............................................................................................82 Open field behavior analysis....................................................................................82 Performance and physiological analysis..................................................................83 Clinical pathology analysis......................................................................................84 Statistical analysis....................................................................................................84 6

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Results.....................................................................................................................................84 Performance.....................................................................................................................84 Behavior..........................................................................................................................84 Physiology.......................................................................................................................85 Discussion...............................................................................................................................87 Conclusion..............................................................................................................................92 6 EFFECT OF FEEDING Mucuna pruriens OR LEVAMISOLE INJECTION ON Haemonchus contortus INFECTION IN LAMBS.................................................................94 Introduction................................................................................................................... ..........94 Materials and Methods...........................................................................................................96 Animals............................................................................................................................96 Treatments..................................................................................................................... ..96 Haemonchus contortus Challenge...................................................................................97 Measurements..................................................................................................................9 8 Statistical Analysis..........................................................................................................9 9 Results...................................................................................................................................100 Clinical Measurements..................................................................................................100 Performance Measurements..........................................................................................101 Discussion.............................................................................................................................103 Conclusion............................................................................................................................107 7 GENERAL SUMMARY, CONCLU SIONS AND RECOMMENDATIONS.....................109 LIST OF REFERENCES.............................................................................................................116 BIOGRAPHICAL SKETCH.......................................................................................................131 7

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LIST OF TABLES Table Page 2-1. Scientific and common names of Mucuna pruriens ...............................................................16 2-2. Ethnobotany: worldwide uses or functions of Mucuna pruriens ...........................................18 2-3. Chemical composition of Mucuna bean meal........................................................................23 2-4. Proximate crude protein concentr ation (g/100 g DM) of beans of 12 Mucuna accessions from Nigeria................................................................................................................... ....24 2-5. Amino acid concentration of Mucuna bean (g/16 g N) and Food and Agriculture Organization/World Health Organization standard values for human diets......................26 2-6. Mineral concentration of different cultivars of Mucuna (mg/100g DM)...............................27 2-7. Fiber fractions in various grain legumes (g/100g DM)..........................................................29 2-8. Antinutritional components of beans of 12 Mucuna accessions from Nigeria.......................30 2-9. Composition of soybean meal and Mucuna beans processed by different methods (DM basis)......................................................................................................................... .........41 3-1. Fermentation characteristics and L-Dopa concentration of Mucuna silage after various ensiling durations...............................................................................................................58 3-2. Chemical composition of unensiled Mucuna (CON) and Mucuna ensiled at various particle sizes for 28 days....................................................................................................5 9 3-3. Fermentation characteristics of unensiled Mucuna (CON) and Mucuna ensiled at various particle sizes for 28 days.......................................................................................60 3-4. Microbial counts and aerobic stability (AS) of unensiled Mucuna (CON) and Mucuna ensiled at various particle sizes for 28 days.......................................................................60 4-1. Effect of processing method on the chemical composition of fine (1 mm) and coarse (6 mm) Mucuna beans............................................................................................................71 5-1. Chemical composition of undetoxi fied (control) and detoxified Mucuna beans...................80 5-2. Effects of feeding unprocessed or detoxified Mucuna pruriens on DM intake and growth of rats................................................................................................................. ....85 5-3. Effects of feeding unprocessed or detoxified Mucuna pruriens on organ weights and concentrations of monocytes, alkaline phosphatase, and phosphorus in the blood...........87 6-1. Effect of feeding Mucuna versus Levamisole injection on performance of lambs..............101 8

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LIST OF FIGURES Figure Page 3-1. Method of ensiling Mucuna beans..........................................................................................53 3-2. The L-Dopa concentration of unensiled and ensiled Mucuna bean......................................57 3-3. Effect of particle size on L-Dopa concentration of ensiled Mucuna ......................................59 4-1. The L-Dopa concentration of fine (1 mm) or coarse (6 mm) Mucuna particles subjected to acid extraction, alkali extraction, or sonication.............................................................70 4-2. Detoxification of Mucuna bean through acid or al kali solvent extraction.............................73 4-3. Color changes afte r detoxification of Mucuna bean...............................................................74 5-1. Experimental conditions for rat feeding trial..........................................................................83 5-2. Effect of feeding detoxified Mucuna pruriens on distance traveled and open field line crossings.............................................................................................................................86 5-3. Effects of feeding detoxified Mucuna pruriens on blood levels of alkaline phosphatase, phosphorus, and monocytes...............................................................................................88 6-1. Experimental conditions for anthelmintic trial.......................................................................97 6-2. Introduction of infectious larv ae from donor goat through gavage........................................98 6-3. Anemia indicators were used to de termine clinical signs of haemonchosis...........................99 6-4. Effect of feeding Mucuna versus feeding a control diet without or with subcutaneous levamisole anthelmintic treatment on fecal egg counts and H. contortus counts in the abomasums...................................................................................................................... .101 6-5. Effect of feeding Mucuna versus feeding a control diet without or with subcutaneous levamisole anthelmintic treatment on packed cell volume and blood protein measurements at va rious time points...............................................................................102 6-6. Effect of feeding Mucuna versus feeding a control diet without or with subcutaneous levamisole anthelmintic treatment on sheep weights at various time points..................103 6-7. Upon necropsy H. contortus worms were harvested from the abomasums and quantified.........................................................................................................................107 9

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DETOXIFICATION, NUTRITIVE VALUE, A ND ANTHELMINTIC PROPERTIES OF Mucuna pruriens By Christiaan Max Huisden August 2008 Chair: Adegbola T. Adesogan Major: Animal Sciences The Mucuna pruriens bean has high protein and starch contents, but also contains 3, 4 dihydroxy-L-phenylalanine (L-Dopa), which has pha rmaceutical properties, but is toxic when ingested by monogastrics. Experiments were conducted to detoxify Mucuna for monogastrics, and to evaluate its anthelmintic effect in rumi nants. Experiment 1a) examined how long it takes to decrease the pH of ensiled Mucuna to < 4.6, the typical minimum pH for ensiled legumes. Crushed beans (6 mm) were ensiled for 0, 3, 7, 21, and 28 days. A pH of 4.5 and an L-Dopa concentration of 1.3% (54% reductio n) were recorded after 28 da ys of ensiling. Experiment 1b) determined the effect of particle size (2, 4, or 6 mm) of ensiled Mucuna on L-Dopa concentration and on fermentation and nutritional characteristics. Ensiling 1or 6-mm particles reduced the LDopa concentration by about 60% while preservi ng most nutrients. Experiment 2 studied the effects of extraction in solutions of acetic acid (ACD, pH 3) or sodium hydroxide (ALK, pH 11) for 8 hours or sonication (SON) for 5 minutes on the L-Dopa concentration and nutritional composition of finely (1 mm) or coarsely (6 mm) ground Mucuna beans. All extraction methods reduced the L-Dopa concentration of fine particles to safe levels (< 0.4%) but increased their neutral detergent fiber (NDF) and starch conc entrations and decreased their water-soluble carbohydrate (WSC) and crude protein (CP) con centrations. Extraction methods were less 10

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effective at reducing the L-Dopa in coarse particles and had inconsisent effects on their nutritional composition. Experiment 3 evalua ted the effect of feeding detoxified Mucuna beans on the performance, behavior, and health of 60 Sp rague-Dawley rats randomly assigned to five treatments. Dietary treatments consisted of a co mmercial rat chow (CON) or diets in which 10% of the rat chow was replaced with either undetoxified Mucuna (MUC), or Mucuna detoxified by ACD or ALK extraction, or ensiling fo r 28 days (SIL). Compared to CON, Mucuna-based diets gave similar feed intake and weight gain. No be havioral abnormalities were caused by any of the diets in open field analyses on days 3 and 10 but when data for both days was collectively analyzed, all Mucuna-based diets exhibited less locomotion than control rats. The decrease in activity was numerically less in rats fed ACD an d SIL diets than in those fed MUC and ALK. Feeding MUC caused splenomegaly and m onocytosis, and reduced blood phosphorus concentrations relative to CON, but detoxification of Mucuna prevented these effects. Experiment 4 determined if ingestion of Mucuna beans reduces helminth parasite infestation in lambs. Thirty-six Dorper x Katahdin ram lambs (28.8 + 5 kg body weight) were randomly allocated to three treatments: a cottons eed meal control diet, a diet in which Mucuna replaced cottonseed meal, and a treatment that involved administering levamisole (2 ml/45.4 kg) to lambs fed the control diet. Diets were formulated to be isonitrogenous (14% CP) and isocaloric (64% total digestible nutrients). Lambs were challenge d 3 times per week for 2 weeks by gavage with infectious H. contortus larvae. Unlike levamisole treatment, Mucuna intake did not affect ( P< 0.05) fecal egg counts (412 vs. 445 eggs/g) or abomasal worm counts (958 vs. 1170 total worms), though a numerical ( P > 0.10) reduction was evident. Neither levamisole nor Mucuna treatment affected anemia indicators, daily feed intake, weight gain or dressing. In conclusion, Mucuna intake did not reduce helminth parasite infection in lambs. 11

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CHAPTER 1 INTRODUCTION Mucuna pruriens is an annual climbing legume indi genous to tropical regions with numerous uses as a food, feed, and nutraceut ical. Like many other legumes, however, it has molecular components that can ad versely affect its nut ritional value, but the ability of these molecules to inhibit enzymes, to selectively bind and enter the ci rculatory system may be useful in pharmacology. Therefore, nutritionists and medi cal researchers have co ntrasting views about the toxicity of the genus. According to Szabo and Tebbett (2 002) the major drawback of Mucuna, which has compromised its usefulness as a food source for either humans or livestock, is associated with its chemical concentration. Mucuna contains novel alkaloids, sap onins, and sterols (Manyam et al., 2004) and a high concentration of L-Dopa. In addition, serotonin and a number of indolic alkaloids structurally related to serotonin have been reported in various parts of the Mucuna plant, several of which have hallucinogenic pr operties of considerable strength (Szabo and Tebbett, 2002). It would, however, be unlikely for these low-level alkaloids to have any effect on human and animal consumers because their abso rption across the gastrointestinal tract is negligible (Szabo, 2003). According to Taylor (2004) beans of Mucuna are not only high in pr otein, but also in nonstructural carbohydrates, lipids, and mi nerals. In South and Mid America, Mucuna beans have been roasted and ground to make a coffee substitute and the bean is also cooked and eaten as a vegetable. Mucuna is also one of the most remarkable green manures as it can add up to 30 ton/ha of organic matter to soils (Pretty et al., 1998). The crop has a long history of use in Indian Ayurvedic medicine and traditional medical practice in several countries where it is used to treat a wide variet y of ailments including 12

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Parkinsons disease (Manyam and Sanchez-Ramo s, 1999; Nagashayana et al., 2001). However, many of such uses are based on anecdotal hea ling properties that require verification and scientific validation. The literature review (Chapter 2) provides an introduction to Mucuna pruriens its taxonomy and characteristics, its use as a food and feed source, its toxic and antinutrient properties, various methods used to detoxify the bean, and Mucunas nutraceutical potential. The research described in subsequent chap ters is aimed at enhancing the use of Mucuna as a food and feed source for monogastrics and to exploit its nutraceutical use as an anthelmintic. Four experiments were conducted. These included de toxification experiments during which ensiling, acid and alkali solvent extractions and sonication were used to reduce the L-Dopa concentration. The primary objectives were to detoxify the bean and to evaluate the nutritional value of the detoxified product (Chapters 3 and 4). In the next experiment, beans produced from three detoxification methods were fed to rats and their behavior, performance, and physiological responses were monitored (Chapter 5). The aim of the final experiment was to determine if incorporation of Mucuna beans in the diet reduces helminth pa rasite infection in lambs (Chapter 6). 13

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CHAPTER 2 LITERATURE REVIEW Introduction Mucuna pruriens is an annual climbing legume that has been used for centuries for a wide array of nutraceutical, nutritional, and other purpose s. It is indigenous to Asia and now grows in many tropical regions, including Africa, South Am erica, and the West Indies. It was grown throughout the southeastern United States as a livestock feed and gr een manure until the 1950s when the advent of cheaper inorganic fertilizer s and soybean meal sources led to its demise (Eilitta and Carsky, 2003). In many tropical countries, Mucuna beans are processed into flour or a coffee substitute, or eaten as a vegetable. Mucuna plants are also used as a highly effective green manure, adding up to 30 ton/ha of organic matter to soils (Pretty et al., 1998). According to Taylor (2004) Mucuna beans are high in starch (39-41%; Ezeagu et al., 2003) and protein (2538%; Ezeagu et al., 2003; Adebowale et al., 2003b). Mucuna like other legumes, contains both beneficial and problematic molecular components. The challenge in promoting Mucuna for human or livestock consumption is identifying cost-effective techniques to reduce its antinutritional properties while preserving its nut rients. The most important antinutrient in Mucuna is 3, 4 dihydroxy-L-phenylalanine (L -Dopa; Szabo, 2003). The beans of most Mucuna species contain a high concentration (2-7%) of L-Dopa. Ingestion of Mucuna has been associated with reduced performance and health in livestock and humans, and most of such problems are thought to result from L-Dopa ingestion. Severa l additional naturally occurring compounds that share tryptamine as a base structure and have ha llucinogenic properties of considerable strength (Szabo, 2003) like serotonin and in dolic alkaloids structurally re lated to serotonin have been reported in various parts of the plant However, it is unlikely for these alkaloids to adversely 14

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affect human or animal consumers because thei r concentrations is low and their absorption across the gastrointestinal tract is negligible (Flore s et al., 2002; Szabo, 2003). Mucuna is used as a treatment for various health problems. There are reports of its use in Indian Ayurvedic medicine as a treatment for worms, dysentery, diarrhea, snakebite, sexual debility, tuberculosis, impotence, rheumatic disorders, muscular pain, gonorrhea, sterility, gout, cancer, delirium, dysmenorrhea, diabetes etc. However, many of these claims derive from common use and have not been verified scientifically. This review focuses on the following aspects of Mucuna pruriens : taxonomy and physical characteristics, nutritional valu e, antinutritional and toxic propert ies, methods for detoxifying the bean and their impact on its nutritional value. Taxonomy and Characteristics of Mucuna pruriens Classification Mucuna pruriens is part of the tribe Phaseoleae and family Fabaceae (Taylor, 2004). Currently the genus accounts for 137 species, subsp ecies or varieties (St-Laurent et al., 2002). The most commonly cited species include M. deeringiana Merrill, M. utilis Wallich (Bengal velvetbean), M. pruriens M. nivea M. Hassjoo (Yokohama velvetbean), M. aterrima Holland (Mauritius and Bourbon velvetbean), M. capitata and M. diabolica (Pretty et al., 1998). Table 21 shows additional species and their common name s. However, the taxonomy of these species has been confused, and some designations may be synonymous (Capo-chichi et al., 2003). More recent taxonomists have considered all cultivars of the velvetbean as Mucuna pruriens variety utilis (St-Laurent et al., 2002). 15

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Table 2-1. Scientific and common names of Mucuna pruriens Scientific Names Common Name Carpopogon pruriens Nescaf Dolichos pruriens Mucuna M. aterrima P de mico M. atropurpurea Fava-coceira M. cochinchinensis Cabeca-de-frade M. cyanosperma Cowage M. deeringiana Cowhage M. esquirolii Cow-itch M. prurita Velvetbean M. utilis Bengal bean Stizolobium aterrimum Mauritius bean S. deeringianum Itchy bean S. pruriens Krame S. pruritum Picapica S. niveum Chiporro Negretia pruriens Buffalo bean Taylor (2004) Species Description The genus Mucuna comprises a species of annual and perennial legumes with vigorously climbing habits that originated in southern China and eastern India, where the plants were widely cultivated as a green vegeta ble crop (Pretty et al., 1998). Mucuna is self-pollinating hence natural out-crossing is rare ; the life cycles range from 100 to 300 days to harvest of the pod and the genus thrives best under wa rm, moist conditions, below 1500 m above sea level, and in areas with plentiful rainfall (Prett y et al., 1998). The Food and Ag riculture Organization (FAO) established climatological growing conditions to be temperatures of 20-30C throughout the growing period with 1200-1500 mm/year or more of rainfall (Bachmann, 2008). According to Taylor (2004), the plant grows 3 to 18 meters in length and its flowers are white to dark purple and hang in long cluste rs or racemes. There are differences among cultivated species in the char acter of the pubescence on the pod (stinging versus non-stinging), bean color (white, beige, mottled, grey, and bl ack), and number of days (110-123 days after 16

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planting) until pod maturity (Pretty et al., 1998; Bachmann, 2008; Chikagwa-Malunga et al., 2008a). Cowitch and cowhage are common English names of Mucuna cultivars whose pods are covered with long reddish-orang e hairs that cause intense irr itation to the skin on contact. The itch is possibly caused by mucu nain, which is a proteolytic enzyme and serotonin, which is a neurotransmitter (Pretty et al ., 1998; Szabo and Tebbett, 2002). According to Taylor (2004), the species name derives from the Latin for itching se nsation and refers to the results of contact with the hairs on the pod. The hair of non-stinging cultivar s, known by the common English name velvetbean are silky and lay flat against the pods surface. Current Uses of Mucuna pruriens Mucuna is used throughout the world for a variety of purposes. For example, it is used as an ornamental crop that forms a dense canopy with visually appealing purple blossoms. Mucuna pruriens is also one of the most popular multi-purpose legumes among small farmers in the tropics because it is an excellent source of green manure, in part because of its ability to fix atmospheric nitrogen (N) thereby restoring soil fe rtility. It also adds si gnificant quantities of organic matter (< 30 tons) to the so il (Gilbert, 2002). Mucunas abundant shallow roots and dense leaves and vines reduce soil erosion, suppress weeds and conserve soil moisture. Nutraceutical Versatility Mucuna pruriens has been used for wide-ranging nutra ceutical applications for centuries. Its beneficial effects are due to its content of pharmacologically active compounds, such as LDopa which is used extensively in the treatmen t of Parkinsons disease. According to Taylor (2004), numerous countries and various cultures claim that Mucuna has various healing properties but most of these ethno-pharmacological claims require scientif ic validation as they are often linked to specific cultural traditi ons and beliefs. Taylor (2004) provided a useful overview of uses of Mucuna in different countries (Table 2-2). 17

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Table 2-2. Ethnobotany: worldwid e uses or functions of Mucuna pruriens Country Uses and ailments or conditions for which Mucuna is used Brazil Anthelmintic, aphrodisiac, diuretic, food, hydropsy, nerve tonic, Parkinsons disease, poison Germany Carminative, cholesterol, hypoten sive, hypoglycemic, muscle pain, rheumatism, rubefacient, anthelmintic India Abortion, alterative, anthelmintic, an tivenin, aphrodisiac, cancer, catarrh, cholera, cough, debility, delirium, diabetes, diarrhea, diuretic, dropsy, dysentery, dysmenorrhea, emmenogogue, fertility, gout, impotency, irritant, lithiasis, nerve tonic, ne rvine, night dreams, scorpion sting, spermatorrhea, sterility, tubercul osis, uterine stimulant, worms Nigeria Antivenin Pakistan Aphrodisiac, diabetes Elsewhere Anasarca, anodyne, anthelmintic, anti dotal, aphrodisiac, asthma, burns, cancer, cholera, cough, cuts, diarrh ea, diuretic, dog bite, dropsy, emmenagogue, insanity, intestinal parasi tes, mumps, nervine, paralysis, pleuritis, resolvent, ringworm, rube facient, snakebite, sores, syphilis, tumors, vermifuge, wind-burns, worms (Houghton and Skari, 1994; Taylor, 2004) Mucuna sap has reportedly been used against pests, as an insect repellent, and its L-Dopa concentration may make it an effective anthel mintic (Faridah Hanum and van der Maesen, 1996). Antioxidant property Some of the espoused healing pr operties are yet to be verified scientifically but research has validated some historical claims. Tripathi and Upadhyay (2002) conducted in vitro and in vivo studies with an alcohol extract of the beans of Mucuna pruriens to investigate its antioxidant properties. The effect was also studied on iron-induced lipid per oxidation, oxidation of glutathione, and its interaction with hydroxyl and superoxide radi cals. There was no change in the rate of aerial oxidation of glutathione but the extract inhibited iron sulfate-induced lipid peroxidation. It also inhibited the specific chemical reactions indu ced by superoxides and hydroxyl radicals. Tripathi and Upadhyay (2002) conc luded that the alcohol extract of the beans 18

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of Mucuna pruriens has an antilipid per oxidation property, which is mediated through the removal of superoxide s and hydroxyl radicals. Antivenin property The Mucuna -based coffee substitute Nescaf has been used as an antivenin and a tonic for nervous system disorders (Pre tty et al., 1998; Taylor, 2004). Mucuna leaf extracts act on thrombin and fibrinogen to enhance blood clotting, which makes such extracts a useful treatment against snake venom-induced hemorrhag e (Houghton and Skari, 1994). Several in vivo studies validate this traditional use. Guerranti et al. ( 2002) demonstrated that the observed antivenin activity has an immune mechanism. Antibodies of mice treated with non-lethal doses of venom reacted against some proteins of Mucuna pruriens extract. Proteins of Echis carinatus venom and Mucuna pruriens extract have at least one epitope in common. The antivenin properties of an extract of Mucuna beans were also demonstrated in vivo by Guerranti et al. (2001). Echis carinatus venom (EV) contains a mixt ure of proteins that inhi bit the coagulative cascade, causing severe bleeding and hemorrhage. The effect of this Mucuna extract on prothrombin activation after EV administration in vitro was studied and an increa se in procoagulant activity was found, potentially explai ning the protective effect in vivo Fertility-enhancing property Misra and Wagner (2007), studied how to best extract L-Dopa from Mucuna for its use in reducing male impotency, as an aphrodisiac, and a nerve tonic. They found that L-Dopa can best be extracted with a 1:1 EtOH-H2O mixture using ascorbic acid as protector, while thin layer chromatography (TLC) fingerprinting may be used to authenticate the pl ant material in the herbal industry. The use of Mucuna as an aphrodisiac was validate d through clinical studies in India. Mucuna increases sexual potency, partly because it increases sperm count and testosterone levels (Siddhuraju et al., 1996). Due to the presence of L-Dopa, Mucuna pruriens can reportedly 19

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be used as an aphrodisiac and prophylactic agen t in patients suffering from oligospermia to elevate sperm count in men and improve ovulati on in women (Lorenzetti et al., 1998; Sridhar and Bhat, 2007). The Mucuna bean also improves sperm motility (Sridhar and Bhat, 2007). Mucunaderived L-Dopa and dopamine are also eff ective inhibitors of prolactin, a hormone released by the pituitary gland th at is considered responsible for 70% of erection failures in males (Vaidya et al., 1978a,b). In one study, oral intake of Mucuna beans in 56 human males improved erection, duration of coitus, and post-coita l satisfaction after 4 w eeks of treatment. Growth-promoting property Mucuna also has anabolic and growth-hormone stimulating properties. The presence of LDopa and thus dopamine in the human system st imulates the release of growth hormone by the pituitary gland (Mesko, 1999). The anabolic effect of the bean is due to its ability to increase testosterone production. In 2002, a U.S. pate nt was filed (Patent No. 6340474; Anon, 2002) on the use of Mucuna pruriens to stimulate the release of growth hormone in humans. Hypoglycemic property Several in vivo studies of Nescafs hypoglycemia-inducing effect validate the traditional use of the Mucuna plant for diabetes treatment (G rover et al., 2001, 2002). Feeding of a Mucuna pruriens seed diet for 1 week to rats reduced fast ing blood glucose levels by 39% (Grover et al., 2002). Plasma glucose concentrations in mice were reduced by 9% when Mucuna was administered (Grover et al., 2001). Furthermore, d ecoction of the leaf (5 g/kg) or bean reduced total cholesterol concentrati on in rats (Taylor, 2004). Anthelmintic property Sources from various countries claim that Mucuna has anthelmintic properties (Taylor, 2004) but there is inadequa te evidence to support this claim. Jalalpure et al. (2007) reported a significant increase in paralysis of worms due to application of a Mucuna pruriens oil extract 20

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Research in which Mucuna was substituted for soybean meal in the ration of sheep (ChikagwaMalunga et al., 2008d) indicated lo wer coccidian oocyst scores ( P < 0.05) and a 52% numerical reduction in fecal egg counts (FEC) in lambs fed a high Mucuna diet relative to a soybean diet. This emphasizes the need for further scientific investigation of the anth elmintic properties of Mucuna. Such studies are particularly important given the increasing problem of parasite resistance to antiparasitic drugs and the increased concern about drug residues in animal products and the environment. These problems make the s earch for biological anthelmintics a priority. The most notorious helminth in tropical and sub-tropical small ruminant production is Haemonchus contortus Haemonchus contortus infects sheep, goats, deer and other ruminants and has been a significant cause of economic loss to small ruminant-producers worldwide (Lange et al., 2006). It is th erefore imperative to examine the anthelmintic effect of Mucuna on this problematic nematode. The considerable egg-laying capacity of H. contortus is maintained by adults feeding on blood. The late stage im mature larvae also feed on blood. Blood loss can result in anemia, anorexia, depression, loss of c ondition, and eventually death of the host animal (Miller and Horohov, 2006). The purported anthelmintic properties of Mucuna require scientific validation. If such claims are proven valid, research would also be needed to id entify the active components to increase the therapeutic use of Mucuna components. Impacts of chroni c ingestion of high levels of Mucuna L-Dopa also warrant further re search (Pretty et al., 1998). Mucuna pruriens as a Food and Feed Source One of the most important uses of Mucuna pruriens is as a source of dietary protein. Dietary Mucuna has played an important role in prev enting malnutrition in Central American countries such as Honduras (Eil itta et al., 2002) and African c ountries such as Benin, Nigeria (Versteeg et al., 1998), Malawi (Gilbert, 2002) and Guinea (Diallo et al., 2002). The use of 21

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Mucuna as a food crop has also been reported in Ghana and Mozambique; during the 18th and 19th centuries, Mucuna was grown widely as a green vegeta ble in the foothills of the eastern Himalayas and in Mauritius, and both green pods and mature beans were boiled and eaten (Sridhar and Bhat, 2007). Mucuna was eventually replaced as a vegetable in Asia by more palatable legumes, but it is genera lly still eaten during famines. In northeastern India it is used as a specialty food. In the mid 1980s, the women of the World Neighbors development program in El Rosario, Honduras used Mucuna to make substitutes for wheat flour, coffee, and cocoa (Bunch, 2002) and developed 22 recipes that were inexpensive, easy to pr epare, highly nutritional, and based on locally available ingredients. The program documented, among other benefits, the positive impact of Mucuna -based nutrichocolate on the milk production of nursing mothers whose breastfed infants progressed in two mont hs from having second-degree malnutrition to no malnutrition at all (Bunch, 2002). In Guatemala and Mexico, Mucuna pruriens has traditionally been roasted and ground to make Nescaf, the main coffee in much of Central America (Pretty et al., 1998; Taylor, 2004). Nevertheless, concerns about the pharmacological properties of compounds in Mucuna have mitigated against wider use of Mucuna as a food source. It is not clear if ingested L-Dopa accumulates in the ti ssues of monogastric livestock consumed by humans, but they do not seem to accumulate in ruminant tissues. ChikagwaMalunga et al. (2008b) fed fort y Rambouillet wether lambs on Mucuna or soybean meal diets and showed that muscle L-Dopa concentrations of all lambs were low and within the normal range (< 5 ng L-Dopa/g), indicating that ingested Mucuna L-Dopa did not accumulate in the animals muscle tissue. The authors conc luded that meat products from sheep fed Mucuna beans 22

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containing about 2% L-Dopa 15 h prior to slau ghter was safe for human consumption. Similar studies are required on monogastric livestock. Nutritional Value Various species of Mucuna are grown as a food crop in many parts of the world because of the nutrient density of the beans (Pretty et al., 1998). Tabl e 2-3 shows the nutrient composition of Mucuna bean meal from Nigeria. Table 2-3. Chemical composition of Mucuna bean meal Parameter Concentration Proximate composition (g/kg DM) Crude protein 354 Crude fiber 77 Ether extract 32 Ash 36 Nitrogen-free extract 479 Major minerals (g/kg DM) Potassium 14 Calcium 10 Magnesium 19 Phosphorus 8 Trace minerals (mg/kg DM) Zinc 13 Manganese 27 Iron 129 Copper 25 Antinutritional factors (mg/kg DM) Hydrocyanic acid 82 Tannins 21 Phytic acid 21 DM=dry matter. Iyayi and Egharevba (1998), Iyayi and Taiwo (2003). Protein Concentration The crude protein (CP) concentration of raw Mucuna bean has been reported to be as low as 21% (Flores et al., 2002) and as high as 38% (Adebowale et al., 2005b). These differences are due to factors like variety, growth environment, and maturity. Pr otein concentration may decline as the bean matures, due to nitrogen availabil ity during bean filling and the final bean size. 23

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Immature beans have been reported to have a c oncentration of 37% CP while mature beans had 24% (Wanjekeche et al., 2003). Tabl e 2-4 shows the difference in CP concentrations of different cultivars of Mucuna. Table 2-4. Proximate crude protein con centration (g/100 g DM) of beans of 12 Mucuna accessions from Nigeria SD=standard deviation. Adapted from Ezeagu et al. (2003). Crude protein M. utilis 29.6 M. cochinchinensis 29.8 M. veracruz (white) 29.4 M. veracruz (mottled) 26.8 M. veracruz (black) 24.5 M. georgia 29.3 M. rajada 29.3 M. ghana 29.2 M. preta 28.0 M. jaspeada 27.6 M. pruriens 27.2 M. deeringiana 27.7 Mean SD 28.2 1.6 The protein concentration of Mucuna beans is similar to that of other food grain legume beans, which can vary from 18 to 44% on a dry weight basis (Kay, 1979). Some studies show that Mucuna protein is made up mainly of albumin and globulins, which typically have a favorable pattern of essentia l amino acids (Bressani, 2002). Because of their high lysine concentration, Mucuna beans are good sources of supplementary protein for monogastric diets based on cereal grains and root crops, which are low in prot ein and lysine (Bressani, 2002; Adebowale et al., 2007). Mucuna contains appreciable amounts of most am ino acids (Ukachukwu et al., 2002), with the exception of sulfur amino acids (SAA). The methionine levels in Mucuna are 1.3 g/16 g N on average, according to Bressani (2002); this value is similar to other beans such as Jack bean (1.2), lima bean (1.5), and 1.2 in pigeon pea (Ukachukwu et al., 2002). The relatively low 24

PAGE 25

methionine concentration of Mucuna is also evident from Table 2-5, which compares the amino acid concentration of Mucuna to the nutritional standards defi ned for human consumption by the Food and Agriculture Organization of the Un ited Nations (FAO) and the World Health Organization (WHO). Supplementation is an effective method for improving Mucunas amino acid profile. The addition of 0.2-0.3% of the limiting SAA typically in creases the protein quality of grain legumes, however digestibility was not improved (Bre ssani, 2002). The usual level of methionine supplementation is 0.3%, if a diet of 10% protein is derived exclusively from cooked beans. This added methionine should raise the total SAA c oncentration of the food to the recommended FAO/WHO level. Mucuna protein digestibility is similar to th at of other grain le gumes (Bressani, 2002). Mucuna beans have an apparent protein digestibi lity ranging from 69-82% in rats (Siddhuraju et al., 1995). Bressani (2002) summarized protein digestibility studies conducted in human subjects and showed that digestibility is a problem in grain legumes due to enzyme-inhibiting polyphenols and the increases in dietary fiber concentration during the cooking of beans. Such polyphenols probably also explain th e variability in the color of Mucuna beans. Colors ranging from white to black reflect differences in protein digestibility. In general, white beans have the highest digestibility (62.1%) followed by black and red Mucuna beans (49.6 and 55.7%), respectively (Bressani, 2002). This is because greater concentrations of the digestibilityinhibiting polyphenolic compounds in the seed coat are present in black or red than in white beans (Sridhar and Bhat, 2007). Although Mucuna is high in protein as well as starch content, it is most valuable as a food or feed when used as a protein source to comp lement cereal-based diets of monogastrics that are 25

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deficient in key amino acids (Adebowale et al., 2007). Diets used to alleviate hunger in developing countries are often characterized by bulky cereal-bas ed porridge which lacks key amino acids (Adebowale et al., 2005a). Mucuna is a particularly useful protein supplement to such diets because its high lysine content compleme nts the lysine deficiency in such cereal-based diets (Bressani, 2002; Adebowale et al., 2007). Table 2-5. Amino acid concentration of Mucuna bean (g/16 g N) and Food and Agriculture Organization/World Health Organization (FAO/WHO) standard values for human diets. Amino acid Average Mucunaa Mucuna utilisb FAO/WHO standardc Lysine 6.6 6.4 5.5 Histidine 3.1 2.2 Arginine 7.2 5.9 Tryptophan 1.4 ND 1.0 Aspartic acid 8.2 8.9 Threonine 3.6 4.2 4.0 Serine 4.1 4.1 Glutamic acid 17.2 14.4 Proline ND 5.3 Glycine 5.1 3.9 Alanine 2.8 3.3 Cystine 0.8 ND Methionine 1.3 1.9 3.53 Valine 5.6 5.3 5.0 Isoleucine 4.1 4.7 4.0 Leucine 7.9 7.2 7.0 Tyrosine 4.7 4.8 Phenylalanine 3.9 5.2 6.0 Adapted from Bressani (2002). ND = not determined; a Kay (1979); b Ravindran and Ravindran (1988); c FAO/WHO reference standard for human amino acid requirements Other Nutrients Mucuna beans are rich in minerals, especially potassium, magnesium, calcium, and iron (Table 2-6; Duke, 1981). Kay (1979) reported that the bean contai ns thiamine and riboflavin at low levels of 13.9 and 1.8 ppm. It contains low amounts of calcium, phosphorus, magnesium and sodium. The relatively high potassium (K) concentration in Mucuna is especially noteworthy. 26

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Iron concentration is of special interest because the diets of many in de veloping countries where Mucuna is grown are deficient in this mineral (Bressani, 2002). The lipid concentration of Mucuna beans varies widely. Some researchers reported low ranges of 2.8-4.9% (Siddhuraju et al., 2000) but others reported hi gher ranges of 8.5-14.0% (Vijayakumari, et al., 2002). Adebowale et al (2 005b) showed that the et her extract of whole seed, cotyledon and seed coat consists of 9.6, 9.8 and 3.0% lipid, respectively. Comparison to Other Legumes According to Bressani (2002), Mucuna beans are similar to common beans and other edible grain legumes in proximate composition, amino acid concentration, micronutrients, content of antinutrients (e.g., en zyme inhibitors, lectins, pheno lic acid, tannin compounds, phytic acid, and sugars). However, only Mucuna, and to a lesser extent V. faba (Faba bean), contain LDopa. Table 2-6. Mineral concentration of different cultivars of Mucuna (mg/100g DM) Mineral Mucuna pruriensa utilisb giganteac pruriensd Sodium 17.4 70.0 35.3 4.1 Potassium 1330.4 1110.0 2295.6 2537.0 Calcium 285.7 250.0 518.3 247.0 Magnesium 85.1 110.0 506.5 72.4 Phosphorus 406.5 220.0 194.3 459.0 Manganese 0.56 1.00 2.36 0.31 Iron 6.54 1.30 9.42 5.19 Copper 2.30 0.60 1.18 0.47 Zinc 2.04 1.00 8.24 1.71 a Siddhuraju et al. (1996); b Ravindran and Ravindran (1988); c Rajaram and Janardhanan (1991); d Mary Josephine and Janardhanan (1992). The lysine concentration of grain legume s ranges from 223 mg/g N for the peanut ( A. hypogaea) to 492 mg/g N in Dolichos ( D. lablab ). The concentration of lysine in the Mucuna bean ranges from 327 to 412 mg/g N (Bressani 2002). Gross energy (GE) ranges from 16.6-17.2 27

PAGE 28

KJ/g (Ezeagu et al., 2003). Mucuna is nutritionally similar to ot her known legume crops, such as Jack bean ( Canavalia ensiformis ) and Yam bean ( Sphenostylis stenocarpa ). It also compares closely with other legumes of West Africa, such as Kidney bean (Phaseolus vulgaris ), Lima bean ( Phaseolus lunatus ), Pigeonpea ( Cajanus cajan), and Bambara nut ( Voandzeia subterranea ) (Bressani, 2002). The nutritive value of Mucuna bean is similar to that of soybean in many respects (Ukachukwu et al., 2002; Kay, 1979). However, important differences also exist; Mucuna has much higher starch content, lowe r fat and protein concentrations and lower SAA concentrations than soybean (Adebowale et al., 2007). The SAA concentration of grain legumes ranges from 96 to 224 mg/g N, and the FAO/WHO reference valu e of SAA in humans for grain legumes is 220 mg/g N. Values of SAA in Mucuna in the literature range fr om 116 to 132 mg/g N (Bressani, 2002), which accounts for only 53-60% of the FAO/ WHO reference value for amino acids in human diets. Therefore, methionine supplementation is advisable when Mucuna is the main dietary protein source. Various studies have shown that consumption of other grain legume beans reduced plasma cholesterol levels and slowly increased blood glucos e levels due to fiber intake from the beans. In general, the acid detergent fiber (ADF) and ne utral detergent fiber ( NDF) concentrations of Mucuna are at least as great as t hose of other grain legumes (Bressani, 2002), as shown in Table 2-7. Therefore, Mucuna bean intake will likely decrease pl asma cholesterol and slowly increase blood glucose levels. 28

PAGE 29

29 Table 2-7. Fiber fractions in va rious grain legumes (g/100g DM) Component Canavalia Cajanus Vigna sp.1 Mucuna ensiformisa cajanb guatemala utilis Acid detergent fiber 11.0 9.1 7.6-7.9 8.9 10.4.6 Neutral detergent fiber 13.6 15.1 9.5-10.7 14.7 20.4.7 Hemicellulose 2.6 6.0 1.6-3.1 5.8 10.0.9 Cellulose 8.1 6.9 5.0-5.7 7.1 9.3.9 Lignin 2.9 2.2 2.2-2.6 1.8 0.8.06 a Bressani and Chon (unpublished data); b Ravindran and Ravindran (1988). Bressani (2002). Antinutritional and Toxic Properties Antinutritional compounds in Mucuna beans include L-Dopa, tanni ns, lectins, phytic acid, and trypsin and amylase inhibi tors (Sridhar and Bhat, 2007). Mucuna is also rich in indolic alkaloids, saponins, and sterols (Manyam et al., 2004). Concentrati ons of these antinutrients in Mucuna are shown in Table 2-8. The stinging hairs of the seed pods contain the phytochemical mucunain, which causes skin irritation and itching (Pretty, 1998). This severe itching could also be due to the serotonin found in Mucuna pods (Szabo, 2003). Because of their common tryptamine base structure and hallucinogenic properties, the indolic alkaloids in Mucuna have also been of concern (S zabo, 2003). Hallucinogenic indoles such as N,N-dimethyltryptamine, bufotenine a nd other tryptamines, including serotonin have been detected in various parts of the Mucuna plant (Daxenbichler et al ., 1972; Lorenzetti et al., 1998). Szabo and Tebbett (2002) measured alkaloids in Mucuna roots, stems, leaves, pods, and bean and reported low concentrations of approximately 0.001%. Serotonin was found only in fresh leaves and stems (~0.001%) and not in the Mucuna bean. However, more recently Szabo (2003), analyzed roots, pods, stems, leaves, and beans for various indolealkylamines and reported indoles to be present at roughly 0.0001% by weight; these levels are lower than previously measured. Many plants contain serotonin including co mmonly eaten fruits such as apples (17 mg/g) and bananas (15 mg/g). By comparison these levels are much higher than those

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30 Table 2-8. Antinutritional components of beans of 12 Mucuna accessions from Nigeria Component M. utilis M. cochinchinensis M. veracruz (white) M. veracruz (mottled) M. veracruz (black) M. georgia M. rajada M. ghana M. preta M. jaspeada M. pruriens M. deeringiana Mean SD L-Dopa (g 100 g-1) 6.82 6.35 5.75 6.43 8.34 7.24 4.00 5.35 7.50 6.57 6.30 8.18 6.57 1.21 Trypsin Inhibitors (TUI/mg) 30.81 42.12 36.97 51.55 36.64 45.97 43.02 38.32 43.27 46.55 45.04 47.63 42.32 5.74 Phytate (g 100 g-1) 0.85 0.79 0.85 0.90 0.26 0.32 0.29 0.53 0.26 0.45 0.37 0.66 0.54 0.25 Phytate-P (g 100 g-1) 0.24 0.10 0.24 0.25 0.07 0.09 0.08 0.15 0.07 0.13 0.11 0.19 0.14 0.07 Phytate-P (as % Total P) 47.89 22.41 49.38 45.26 14.18 16.57 18.43 33.79 14.57 27.10 24.44 34.38 29.03 12.97 Total Oxalate (mg 100 g-1) 1.35 2.48 2.08 1.98 1.13 1.53 1.98 2.95 2.31 1.81 1.26 2.85 1.98 0.60 Soluble Oxalate (mg 100 g-1) 1.12 1.19 1.71 1.08 1.08 1.08 1.76 1.89 1.85 1.08 1.17 2.01 1.42 0.38 Soluble oxalate (% total) 82.96 78.63 82.21 54.55 95.58 70.59 88.89 64.07 80.09 91.53 92.86 70.53 79.37 12.47 Cyanide (mg 100 g-1) 0.12 0.12 0.12 0.10 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.01 Nitrate-N (mg 100 g-1) 1.80 ND ND 1.60 ND ND ND ND ND ND 5.20 2.40 2.75 1.67 Tannin (g 100 g-1) 1.62 1.63 1.62 1.60 1.59 1.63 1.66 1.70 1.65 1.57 1.62 1.56 1.62 0.04 The values are means of two independent determin ations. ND=Not Detected. Ezeagu et al., 2003.

PAGE 31

reported in Mucuna bean (Szabo and Tebbett, 2002). Most tryptamine derivatives are also characterized by poor absorption and rapid peripheral metabolism; thus the presence of low level indolealkylamines is unlikely to affect the use of Mucuna as a food and feed source (Szabo, 2003). Tannins It has been reported that ta nnic acid consumption negatively impacts the protein efficiency ratio and apparent protein digestibility of red P. vulgaris (Bressani, 2002); as intake of tannins increases, protein quality and protein digest ibility decreases, therefore the tannins in Mucuna may affect its viability as a protein source. Tannins are natural defense compounds found in plants that make the plant unpalatable due to their astringency. Tannins, like other phenolic compounds, vary widely in type, concentration and astringency. In addition to these factors, tannin-bin ding effects depend on animal physiological status, sex, exposure to pathoge ns, anticipated productive performance and environmental conditions (Waghorn et al., 2003). Th e structural diversity of tannins within and between plant species causes variat ion in their biol ogical activity. According to Reed (1995), tannins are water-soluble polymeric phenolics that form strong complexes with proteins. The strength of thes e complexes depends on characteristics of both tannin and protein (molecular weight, tertiary stru cture, iso-electric point, and compatibility of binding sites; Waghorn et al., 2003). Horvath (1981) gave a more comprehensive definition that reflects the fact that tannins form complexes with starch, cellulose, minera ls as well as protein. The antinutrient activity of tannins starts ea rly in the digestive tract where they form complexes with salivary glycoproteins resulting in reduced feed intake. This tanninmucoprotein complex diminishes the lubricant property of saliva, leading to dryness of the oral cavity. Tannins reduce feed digestibility by binding bact erial enzymes and forming complexes with cell 31

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wall carbohydrates; they also ex ert inhibitory effects on the growth and activity of rumen microbes and can be carcinogenic (Wang et al ., 1976). The two major cl asses of tannins are condensed tannins or proanthocyanidins (PA) and hydrolyzable tannins (HT; Reed, 1994). Proanthocyanidins Proanthocyanidins, the most co mmon type of tannin found in forage legumes, have been considered non-toxic; however, th ey are associated with lesions of the gut mucosa and can result in extensive binding of proteins, including microbe s, salivary, and enzyme sources (Waghorn et al., 2003). Proanthocyanidins are fl avonoid polymers confined to in tracellular vacuoles and are essentially un-reactive until released by cell rupt ure (Waghorn et al., 2003). Tannins reduce cell wall digestibility by binding bacterial enzymes and forming indigestible complexes with cell wall carbohydrates. Digestibility of organic matter and fiber frac tions is low for high PA diets (Reed, 1994). Chewing initiates binding betw een PA and plant and saliva ry proteins causing protein aggregation instead of the norma l solubilization. Proanthocyanidins are indiscriminate in the protein to which they bind and easily create a protein (N) deficiency, especially when poorquality grasses make up a substantial portion of the diet (Waghorn et al., 2003). Hydrolyzable tannins Hydrolyzable tannins are especially toxic to ruminants. Pyrogallol, a hepatotoxin and nephrotoxin, is a product of HT degradation by ru minal microbes. Hervas et al. (2002) reported striking lesions in the digestive tract, distensi on of abomasum and small intestine, and dense mucous material in the caecum, and changes in plasma biochemistry in sheep fed high (3 g quebracho tannin extract/kg live-w eight) tannin levels. The major lesions associated with HT poisoning are hemorrhagic gastroenteritis, necr osis of the liver, and kidney damage with proximal tubular necrosis. 32

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Tannins can be used to increas e post-ruminal protein availabil ity in ruminants. Preston and Leng (1990) demonstrated that inclusion of tanni ns in low amounts in ruminant diets reduced ruminal fermentation and allowed protein to bypass the rumen to the lower intestine where it was more readily available. However, the release of the protein post-ruminally depends on the nature of the tannin-protein complex and the prevailing pH ( Salawu et al.,1999) The Mucuna plant contains up to 8% tannins, mostly concentrated in the leaves (4.4-7.4%; Chikagwa-Malunga et al., 2008a); su ch levels can reduce DM digestibility and protein utilization (Makkar et al., 1988; Mangan, 1988; Orskov and Miller, 1988; Dalzell et al., 1998). As shown in Table 2-8, the bean regularly contains lower conc entrations of tannins (1.6-1.7%; Ezeagu et al., 2003). These levels may be beneficial in ruminant diets since they can promote the bypassing of the rumen. Levels considered beneficial for th is purpose range from 2-4% of DM while higher levels have been associated with reduced digestibility in livestock (Mueller-Harvey and McAllan, 1992). Although th e concentrations in Mucuna beans and pods are reportedly low and the majority of tannins in legumes are non-toxic proanthocyanidins, more research is needed to ascertain the hydrolysable tannin content of Mucuna beans. L-Dopa The beans of most Mucuna species contain relatively high concentrations of 3, 4 dihydroxy-L-phenylalanine (L-Dopa), the aromatic amino acid precursor of the neurotransmitter dopamine. Mucuna beans from various sources and cultivars contain 2.3-7.6% L-Dopa (StLaurent et al., 2002); this is in agreement with the reported ra nge of 3.1-6.7% by Daxenbichler et al. (1972). However, one study reported as little as 1.5% L-Dopa in the bean of M. gigantea in southern India (Rajaram and Janardhanan, 1991). The wide range and discrepancies may reflect the improper taxonomy of Mucuna pruriens cultivars, though variations among cultivars may 33

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also be due to genotypic variati on, maturity, latitude, and envir onmental factors (St-Laurent et al., 2002). Due to the abundance and toxic nature of LDopa, it is of greate r concern than other antinutrient components of Mucuna (Szabo, 2003). The consequences of consuming toxic levels of these compounds can be severe. For instance, an outbreak of acute psychosis in Mozambique was attributed to excessive consumption of velvetbean L-Dopa (Infante et al., 1990). This resulted because, during a famine and drought, the water used to boil and detoxify the bean was consumed rather than discarded as it normally is and thus a higher than normal volume of the toxins was ingested (Pretty et al., 1998). In one study, L-Dopa was assayed in the roots, stems, leaves, and pods of dried Mucuna pruriens, variety utilis plants, the stems and leaves of fresh plant material, in raw bean samples, and in bean prepared according to four different recipes (Szabo and Tebbett, 2002). Concentrations were 0.15% in dried leaves and pods, 0.49% in dried stems 4.47 to 5.39% in the raw bean, 0.10% in beans boiled repetitively, and 2.38% in roasted beans. Chikagwa-Malunga (2008a), reported that L-Dopa concentrations in the stems ( 0.1-0.2%) and whole plant (0.1-1.8%) peaked at 110 days after planting while the conc entration in the leaves remained constant (0.10.3%) during plant maturation of Mucuna pruriens, variety utilis Whole pods contained up to 4% L-Dopa and this concentration decreased with maturity. It is evident from the literature that the highest concentration of L-Dopa is present in the seed, which is the main part of the plant used in monogastric diets. Pharmacodynamics of L-Dopa in humans Most of the adverse effects of Mucuna consumption by humans ar e associated with LDopa, which is an intermediary product in the enzymatic synthesis of dopamine from L-tyrosine (Szabo and Tebbett, 2002). Dopamine regulates f unctions in the brain (neurotransmitter), heart 34

PAGE 35

(inotropic increase of car diac output), vascular system (vesse l dilator), and kidney (diuretic) (Grossman et al., 1999; Grover et al., 2001). L-Dopa is widely di stributed in muscle, kidney and liver, and present across the blood-brain barrier in the central nervous system due to de novo synthesis (Szabo and Tebbett, 2002). Many of these physiological e ffects are correlated with the de novo synthesis and level of intake of L-Dopa. Szabo and Tebbett (2002) provided the following description of th e pharmacokinetics in humans: Approximately 33% of an orally admini stered dose of L-Dopa is absorbed from the human gastrointestinal tract, primarily from th e jejunum. Peak plasma concentrations occur within 1 to 3 hours of ingestion with levels that may vary as much as tenfold among individuals. L-Dopa undergoes decarboxylation into dopamine extr aand intracerebrall y. A majority of the successfully absorbed L-Dopa is co nverted to dopamine in the periphery, mainly in the intestinal mucosa via decarboxylation by the enzyme L-ar omatic amino acid decarboxylase (LAAD). In addition to dopamine, peripheral L-Dopa is metabolized to melanin, norepinephrine, 3methoxytyramine, methyldopa, 3,4-dihydr oxyphenylacetic acid, and 3-methoxy-4hydroxyphenylacetic acid (homovanillic acid or HVA). These metabolites are rapidly excreted in the urine such that about 80% of an administered dose of L-Dopa is excreted within 24 hours; less than 1% of which consis ts of the original compound (D ollery, 1999; Szabo and Tebbett, 2002). Of the L-Dopa metabolites in the urine, roughly 50% consist of dihydroxyphenylacetic acid (DOPAC) and HVA, 10% of dopamine, while less than 30% occurs as 3-O-methyldopa (Dollery, 1999). Dopamine produced by decarboxylation of L-D opa by LAAD in the periphery does not cross the blood-brain barrier, but is further metabolized by mono amine oxidase (MAO) in endothelial cells into DOPAC (Szabo and Tebbett, 2002). Less than 1% of the administered dose 35

PAGE 36

actually crosses the blood-brain barrier into the central nervous system and the basal ganglia. In the brains basal ganglia it is converted to dopamine by LAAD or dopa decarboxylase. Dopamine subsequently undergoes enzymatic in activation catalyzed by MAO and catechol-O methyltransferase (COMT). The COMT in the glial cells, methylate dopamine to 3methoxytyramine, while MAO oxidizes dopamine to DOPAC. The COMT also methylates about 10% of oral L-Dopa to 3-O-methyldopa in the red blood cells and in th e liver (Dollery, 1999). The COMT and MAO together convert dopamine to 3-methoxy-4-hydroxyphenyl acetic acid and HVA. Adverse effects of L-Dopa on humans Most side effects of L-Dopa ingestion in hum ans arise directly from dopamines activity as a neurotransmitter involved in the regulation of the heart, vascul ar system, digestive tract, and excretory system, rather than from its wellknown effect on receptors in the brain. Some consequences of increased peripheral dopamine include orthostatic hypotension resulting in dizziness and in some cases staggering and in creased heart rate (S zabo and Tebbett, 2002). Although tolerance to the effects develops in in dividuals ingesting L-D opa over a few months, the heart-related effects could pose a serious problem for those who have a predisposing cardiac condition (Szabo and Tebbett, 2002). In contrast, some studies report improved cardiac function after L-Dopa administration to patients with re fractory or congestive heart failure (Grossman et al., 1999). This was because oral L-Dopa treatment produced bene ficial natriuretic and diuretic effects because the resulting dopamine increases renal blood flow and increases the force of cardiac contraction. These beneficial effects temporarily relieve the symptoms and signs of heart failure. Psychiatric disturbances have also been repo rted in patients receiving high doses of LDopa in a dose-dependent manner (Szabo and Tebbett, 2002). These include mild nervousness, 36

PAGE 37

anxiety and agitation, insomnia, and vivid dreams. About 15% of Parkinsons patients have experienced serious psychiatric manifestations as a result of L-Dopa therapy. Confusion, delirium, depression and, in extreme cases, psycho tic reactions with hallucinations have been reported (Szabo and Tebbett, 2002), suggesting that L-Dopa should be avoided by patients with a history of psychosis or epilepsy. Also, due to its inotropic and vasodilating activity, L-Dopa should be avoided by patients with glaucoma, asthma, renal, hepatic, cardiovascular or pulmonary disease (Dollery, 1999). Si nce it is a precursor of mela nin, which is associated with melanoma formation, L-Dopa could promote the formation of skin cancers (Dollery, 1999). Based on anecdotal evidence, the Mucuna bean could potentially cau se contraction of the womb (Taylor, 2004); likely due to its inotropic property and this could be useful in inducing or assisting during labor. However, Mucunas usefulness as a uterine stim ulant is jeopardized by its potentially harmful impact on the unborn baby. Szabo and Tebbett (2002) report that L-Dopa consumed by nursing mothers can also appear in breast milk, causing harm ful side effects in infants. Nausea, vomiting, and anorexia have b een reported as side effects of Mucuna ingestion, and are associated with the effects of periphe rally produced dopamine on the dopamine receptors in the area postrene of the brain stem. Naus ea and vomiting may occur on consumption of as little as 250 mg of L-Dopa if the patient is unaccustomed to such an exposure (Szabo and Tebbett, 2002). These adverse effects em phasize the need for detoxification of Mucuna before it is fed to humans. Adverse effects of L-Dopa on monogastric livestock As in humans, most adverse effects of Mucuna intake in monogastric livestock are attributable to its L-Dopa con centration. Symptoms in broilers and pigs include reduced feed intake, weight loss, and low feed conversion (Del Carmen et al., 2002; Flores et al., 2002). In one 37

PAGE 38

study, Flores et al. (2002) reported reduced palatability, daily weight gains, feed intake, and feed conversion with substitution of Mucuna beans for soybean meal in pig diets. Necropsy revealed acute toxic hepatitis and advanced necrosis in the pigs fed the Mucuna bean. Liver failure was also evident as blood analysis showed abnormal c oncentrations of glutamic pyruvic transferase, aspartate amino transf erase, and bilirubin. Relative to control animals, abnormalities have been observed in animal growth, carcass and individual organ weight, a nd blood plasma levels of tr iiodothyronine, cholesterol and creatine in chickens fed raw Mucuna beans (Carew et al., 2002; Del Carmen et al., 1999). According to Del Carmen et al. (1999), reductio n in growth and carcass weight of broilers resulted from inclusion of 10 30% of raw Mucuna in the diet. Feed inta ke decreased at the 30% inclusion rate while feed efficiency fell at both 20 and 30% rates. Feed intake was less severely affected than growth, indicating that reduced fe ed intake was not the major cause of growth decrease. Rather, the au thors suggested that Mucuna bean inhibited metabolic processes, leading to growth reduction. This was in agreement with Carew et al. (2002), who observed similar reductions in growth and feed intake when Mucuna levels were increased in chick diets. Additionally, Carew et al. (2002) reported reduced growth, increased wei ght of the gizzard and pancreas, and lengthening of the small intestine and ceca in chicks fed raw Mucuna beans. The increased gizzard and pancreatic weight were attributed to Mucunas poor digestibility and trypsin-inhibitor concentr ation, which increased the demand on these organs to secrete more exocrine digestive enzymes. Low concentratio ns of plasma triiodothyronine (T3), blood cholesterol and plasma creatine were also associated with Mucuna bean intake, and these reflected increased glomerular filtration rate in the kidney, as well as low muscle mass. The concentration of the cytoplasmic enzyme, alanine aminotransferase increased with Mucuna bean 38

PAGE 39

intake, indicating either liver or muscle damage Decreased plasma concentrations of creatine provided strong evidence of muscle damage but al kaline phosphatase, anot her indicator of liver damage, did not confirm the damage in chickens. The foregoing emphasizes the need to investigate methods of reducing the L-Dopa concentration of Mucuna to safe levels. Safe levels of dietary Mucuna L-Dopa The dose at which a chemical becomes toxic is highly dependent on the prevailing conditions and the applications; compounds that are considered toxic at certain levels of exposure may help sustain, improve, or restore he alth at other dosages. All chemicals can be considered toxic if the dose is high enough and ev en highly toxic chemicals may be used safely if exposure is kept low enough (Williams et al., 2000). Mucuna L-Dopa has long been used in the treatment of Parkinsons disease (Manyam and Sanchez-Ramos, 1999; Nagashayana et al., 2001) and other ailments (Taylor, 2004), yet the toxicity of Mucuna L-Dopa has limited its use for dietary purposes (Szabo and Tebbett, 2002). Szabo and Tebbett (2002) suggested that Mucuna L-Dopa can be transf erred to milk after consumption of Mucuna-based products. However, recent research indicates that ingested Mucuna L-Dopa does not accumulate in ruminant tis sues (Chikagwa-Malunga et al., 2008b). Published studies on accumulation of Mucuna L-Dopa in monogastric tissues were not found. Therefore, although guidelines on safe Mucuna L-Dopa concentrations for monogastric diets exist, it is unclear if ingestion of L-Dopa at such levels result s in L-Dopa accumulation in the tissues of monogastric livestock consumed by humans. Guidelines on safe Mucuna L-Dopa concentrations for m onogastric diets are based on in vivo experiments (Carew et al., 2003; Ferriera et al., 2003; Iyayi and Taiwo, 2003; Ukachukwu and Szabo, 2003) as well as data gathered from use of L-Dopa in the treatment of Parkinsons Disease (Szabo and Tebbett, 2002). Several resear chers have agreed that L-Dopa concentrations 39

PAGE 40

of 0.3 0.4% of Mucuna (DM basis) are safe for monogastri cs based on multiple studies with poultry (Eilitta et al., 2003). However, this safe limit for poultry should be considered a guideline for other monogastric species because the level of L-Dopa toxicity probably varies with animal species, metabolic rate, performance level, body and health condition, and the level of intake relative to body weight. According to Lorenzetti et al. (1998), the maximum daily dose of Mucuna L-Dopa that can be tolerated by an adult person without causing side effects is approximately 1500 mg per day. Therefore, a healthy adult should be able to safely consume 500 g of Mucuna-based food containing 0.1% L-Dopa, and dietary Mucuna or Mucuna products should contain no more than 0.1% L-Dopa. However, this guideline may not apply to long-term L-Dopa ingestion or to Mucuna consumption by children, pregnant women, a nd people with a medical condition (Szabo and Tebbett, 2002). This safety level is in agreem ent with Teixeira et al. (2003) who concluded that 0.1% L-Dopa is an acceptable target level, based on the fact that Faba bean and Broad bean (Vicia faba) contain 0.2 0.5% L-Dopa, yet they have b een safely consumed for generations by people around the world. Although these safety targets are invaluable, future research should focus on obtaining more specific guidelines that acc ount for species and physiological stage. In order to determine the dose-response relationship of Mucuna L-Dopa on monogastrics, de tailed toxicity testing is required for a significant duration of time. The response to these dosages should range from a lethal dose (e.g. LD50) to a dose that shows no observable eff ect levels (NOEL) or side effects under the established time frame. Using establishe d rules of risk assessment, these findings can then be extrapolated to other species, including the human (Williams et al., 2002). 40

PAGE 41

Detoxification of Mucuna pruriens Processing methods have been developed to facilitate nutrient util ization of many food grain legumes for both human and animal c onsumption. Table 2-9 shows the nutritional concentration of soybean and Mucuna processed by some of such pr ocedures. In the case of Mucuna, processing methods can serve the additi onal purpose of detoxifying the bean by decreasing the L-Dopa concentration to a safe level while maintaining its nutritional benefits (Bressani, 2002). Some of the processing methods used with Mucuna include soaking in water, alkalis or acids, and various cooking methods including dry heating (roasting), boiling and frying, as well as germinating or fermenting. Th ese processing methods are discussed below. Table 2-9. Composition of soybean meal and Mucuna beans processed by different methods (DM basis). Meal DM % CP % CF % Ash % Ca % P % L-Dopa % Soybean meal 90.15 48.00 6.28 5.73 0.25 0.60 NA Toasted Mucuna 92.65 22.31 6.16 3.48 0.14 0.35 2.76 Cooked Mucuna 88.45 22.10 5.63 3.16 0.14 0.36 2.58 Soaked Mucuna 89.16 21.9 6.16 3.27 0.16 0.36 2.55 Raw Mucuna 88.98 21.77 4.69 3.17 0.17 0.42 3.99 DM: Dry Matter; CP: Crude Prot ein; CF: Crude Fiber; Ca: Ca lcium; P: Phosphorus; NA: not applicable; Toasted = dry heated for 30 min. at 130oC; Cooked = boiled for 30 min.; Soaked = immersed in water at room temperature for 48 hours; Raw = untreated. Flores et al. (2002) Thermal processing methods include cooking bean s at atmospheric pressure or pressurecooking, without or with previous soaking in water, which typically reduces cooking time. Although such methods can reduce L-Dopa con centrations, cooking increases dietary fiber concentration from approximately 19 to 26% an d the fiber traps protein and probably makes it unavailable (Bressani, 2002). Cooki ng also induces losses of vitamins (25-30%) and minerals (10-15%). Specific thermal processing methods include the following: 41

PAGE 42

Boiling According to Teixeira et al. ( 2003), L-Dopa extraction from ground Mucuna beans (1 mm particle size) can be achieved by increasing the water temperatur e. The extraction time required for reducing the L-Dopa concentration to 0.1% can be reduced from 55 hours at 20oC to < 1 hour at 100oC. Attempts to replace boiling in water by extr action with acidic or alkaline solvents or to reduce boiling time by presoaking before boiling have yielded different resu lts. Nyirenda et al. (2003) soaked Mucuna grits (4 mm particle size) in wate r with and without sodium bicarbonate for 24 hours, and then they boiled it for 1 hour and soaked it again for 24 hours in order to achieve an 88% L-Dopa reduction (0.4% L-Dopa). They concluded that boiling alone was the principal method for eliminating L-Dopa and th at the soaking steps may not be worthwhile. Ukachukwu and Szabo (2003) used we t heating (boiling for 30-45 min) of beans, with or without additives (4% wood ash, sodium carbonate or calcium hydroxide) to detoxify Mucuna and tested the product in broiler diets. Broilers fed on b eans previously treated with wood ash and boiled for 45 minutes performed best and such treated bean s could be included at levels of up to 30% of the ration of broilers without causing adverse e ffects on weight gain, f eed conversion ratio, and protein efficiency ratio relative to a maize-soybean dietary cont rol group. Nyirenda et al. (2003) fed broilers on diets in which 50% of the dietary CP was from Mucuna beans detoxified through boiling for 60 minutes and subsequently drying at 50oC for 18 hours or soaking in 0.25% and 0.50% baking soda for 24 hours. They found that th e rations produced similar feed intake, weight gains, and feed conversion ratios as cont rol diets consisting of maize and soybean. When Ukachukwu and Obioha (1997) compared the efficacy of dry and wet thermal processing methods, such as toas ting (dry heating) or boiling in water at a temperature of 100oC, they found that boiling was more effective as it reduced hydrocyanic acid concentration by 25%, 42

PAGE 43

hemagglutinin activity by 50%, trypsin inhibito r activity by 43%, tannin concentration by 10%, and L-Dopa concentration by 31-41% (Ukachukwu and Obioha, 2000). Roasting Roasting beans enhances the flavor and reduc es the L-Dopa concen tration up to 45% but reduces their nutritive value by lowering the leve ls of available lysine and other amino acids (Bressani, 2002). Heating at 130oC for 30 minutes reversed many adverse effects of L-Dopa on blood chemistry and anatomy in poultry. Inclusion of 10% roasted Mucuna beans in the diet of chickens resulted in better growth and carcass yield relative to a raw Mucuna treatment since trypsin inhibitor activity was e liminated by heating (Del Carmen et al., 1999). Heating also eliminated amylase inhibitor activity. Iyayi and Taiwo (2003) reported that laye rs fed 6% of the diet as roasted Mucuna beans maintained good egg quality relative to a soybean-based control diet. The beans were roasted with sand over an open fire for about 40 minutes till their shiny seed co ats became dull. They also reported that replacing 33% of dietary soybean meal with Mucuna beans did not reduce feed intake or weight gain but did cause kidney dama ge. From an animal production standpoint, this 33% replacement may seem acceptable since it ma y not negatively affect the production and economic gain during the animals productive life. From an animal health standpoint, however, the adverse changes in physiology are important si nce they can cause unnecessary discomfort to the animal. Further research is needed to determine the appropriate incl usion rate of roasted Mucuna beans for maintaining animal health. Diallo et al. (2002), report th at the most effective detoxifi cation method was to roast the beans for 20-30 minutes, crack them, leave them in water for 48 hours while changing fresh water every 8-12 hours, and then cook them for about 2 hours. This technique removed L-Dopa to a level below 0.1% but it is more labor intensive than many others. 43

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Of the processing methods mentioned thus fa r, only those methods that utilized boiling decreased the L-Dopa con centration to or near the target level. While roasting removed about half the L-Dopa, permeation of the seed body with hot water was necessary to extract >75% of this water-soluble compound. Therefore, boiling is preferable to roasting for L-Dopa removal. Although many thermal processing methods are e ffective in reducing L-Dopa levels and rendering the bean safe for monogastric consump tion, applying sufficient heat requires time, energy and expense. Many energy sources for rapidly heating Mucuna would be costly or unavailable in develo ping countries where Mucuna consumption is likely to be most beneficial. Therefore it is important to investigate alternative methods. Ensiling Mucuna has been fermented with Rhizopus oligosporus to make food products such as tempe (Egounlety, 2003). The fermentation did not reduce CP concentrat ion after 48 hours but increased the concentration of water-solubl e proteins from 1.22% to 19.42%, indicating increased proteolysis. Matenga et al. (2003) studied effects of ensiling different mixtures of maize and Mucuna and reported a reduction in L-D opa content of 10% for a 100% Mucuna with 0% maize mixture, and a reduction of 48% for a 30% Mucuna with 70% maize mixture. Therefore, fermentation might be a useful Mucuna detoxification strate gy and the efficacy seemed to increase as more fermentable carbohydrat es were supplied in the mixture. However, no other studies examining effects of fermenting Mucuna by itself or without supplemental micro organisms were found in the literatur e. Therefore this method of detoxifying Mucuna requires further study. Solvent Extraction Solvent extraction is widely used in the food, pharmaceutical and chemical industries to extract a soluble constituent from a solid. Solvent extraction involves three steps: (1) submerging 44

PAGE 45

the solute in the solvent; (2) separating the solution formed from the spent residue; and (3) washing the spent residue (Bal aban and Teixeira, 2002). High te mperatures can be used to facilitate this process. However, for the extraction to be successful, the beans must be broken into fragments and must be in contact with the water for a sufficient amount of time. Solubility in water Water is the most readily available solvent for smallholders, but according to the Merck Index (1983), L-Dopa has only limited solubility in water (66 mg in 40 mL). Assuming an initial concentration of L-Dopa in Mucuna bean of 6-7% dry weight, this translates into the need for 40 parts of water to one part bean by weight (40 li ters of water per kg of beans; Teixeira et al., 2003). This may be unrealistic for smallholders in places where copious amounts of water are not readily available or where clean water is expensive. Nyirenda et al. (2003) soaked Mucuna grits (4 mm particle size) in water for 24 hours (1:1 ratio by weight of water to bean), they then boi led it for 1 hour and soaked it again. They came to the conclusion that boiling was effective at re ducing L-Dopa but the soaking steps were not. Their observation was likely based on the fact that not enough water was used to satisfy the solubility requirements and that th e particle size might have been insufficiently small. Teixeira et al. (2003), observed a reduction in L-Dopa concentration to below 1% within 24 hours of extraction in water alone for 1mm but not in 2-, 4-, or 8-mm Mucuna bean particles. They used a 28 L water bath to submerge 45 five gram sa mples under continuous wate r circulation of 15 L per minute. Therefore, extraction rates can be quite high provided 1) sufficient water is used, 2) the Mucuna bean is ground into a sufficiently small pa rticle size, and 3) agitation is performed. Two solvent extraction processing methods using water and technology more readily available to smallholders in developing countri es with access to rudime ntary food preparation tools are the Sack in Stream and Overflowing Trough and Rake methods (Diallo et al., 2002). 45

PAGE 46

Sack in Stream In the Sack and Stream method, a strong, porous sack filled with cracked and de-hulled Mucuna beans is submerged in a flowing stream or river (Diallo et al., 2002). This method brings a constant supply of continuous-flowing pure solvent in contact with the bean particles with little energy, labor, or equipment required. The force of the water flowing through the sack enhances L-Dopa extraction at the particle /solvent interface, and the constant supply of fresh water maintains a maximum concentration gradie nt for effective extraction. Immersing Mucuna beans within a porous bag in a flowi ng river for 3 days reduced L-Dopa concentration of cracked and whole beans to 0.2 and 0.72%, respectively (Dia llo and Berhe, 2003). Depending on the size and flow rate of the river and the amount of L-Dopa released from the beans, adverse impacts on marine life could occur. Long-term damage woul d be unlikely however, gi ven the shor t half life (1 hour) of L-Dopa (Murata, 2006), modest Mucuna volumes processed by smallholders, and the low concentrations of L-Dopa due to constant dilution. Overflowing Trough and Rake This method involves submerging cracked, de-hulled Mucuna beans in a watering trough or other such vessel, and filling it to overflowing while stirring the beans with a rake (Diallo et al., 2002). When extraction is completed, the trough is drained, and the beans recovered for use. This method allows for relative velocity at the particle/solvent interface as well as for agitation, both of which are required for proper extraction (Teixeira et al., 2003). Ho wever, the constant need for fresh water and the labor intensive sti rring would limit its adoption. The efficacy of the method at reducing L-Dopa has not been studied. The procedure n eeds to be standardized with respect to particle size, water temperature, in tensity and duration of agitation, and amount and velocity of the water supply. 46

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Solubility in alkaline and acid solvents Alkaline conditions may facilitate the inactivation of L-Dopa in Mucuna beans (Wanjekeche et al., 2003). Experiments ha ve demonstrated that soaking cracked Mucuna beans in a solution of lye (calcium hydroxide, Ca(OH)2) reduced the concentrat ion of L-Dopa to less than 0.01% (Diallo et al., 2002). However, the treat ed material was remarkably dark in color; therefore further research was advocated to determ ine its acceptability to humans. Diallo et al. (2002) indicated that soaking Mucuna beans in 4% Ca (OH)2 solution for 48 hours effectively detoxified the bean to a level of 0.001% L-Dopa. This is in agreem ent with Teixeira et al. (2003), who reported that a NaOH solution extraction of Mucuna beans (1-mm particle size) at pH 11 reduced the L-Dopa concentration to < 0.1% in less than 8 hours. However, these authors also noted that the consistently dark color of the al kali-extracted bean may reduce its acceptability to consumers. This dark coloration is due to production of melanin from the L-Dopa (Latellier et al., 1999; Teixeira et al., 2003; Wanjekeche et al., 2003). L-Dopa is also readily soluble in dilute solutions of acetic acid (Teixeira et al., 2003). In fact, most traditional laboratory analytical me thods for quantifying L-Dopa begin with extraction in hydrochloric acid (Daxenbichler et al., 1972). Soaking Mucuna beans in acidified water (pH 3) reduced the L-Dopa in Mucuna beans (1-mm particle size) to safe levels (<0.4 mm) in less than 8 hours (Teixeira et al., 2003). According to Wanjekeche (2003), beans cooked in acid solutions are darker than raw beans but lighter th an beans cooked in alkaline solution. This is presumably due to less melanin formation. Siddhuraju and Becker (2005), soaked cracked Mucuna beans for 24 hours at room temperature in either 0.07% s odium bicarbonate or 0.1% ascorbic acid before autoclaving the beans for 20 minutes at 121oC. The L-Dopa content was redu ced to 1.2 and 1.5% in the alkaline and acidic solutions respectively. It is, however, not certain how much of this effect was due to 47

PAGE 48

the solvent extraction since the extracted beans were autoclaved before analysis, and pressurecooking is one of the most effective methods of L-Dopa removal (Bressani, 2002; Teixeira et al., 2003; Nyirenda et al., 2003). Therefor e, research is still needed to investigate if acid extraction is more effective than alkaline extraction, and to determine the nutritional value of the extracted beans. Sonication Agitation by stirring, raking, or shaking is effective at part ially reducing L-Dopa levels (Diallo et al., 2002; Teixeira et al., 2003) but St-Laurent et al. (2002) reported complete extraction of L-Dopa from Mucuna in less than 5 minutes by pl acing 0.1 g of the powdered bean in 15 g of distilled water (150 parts water to 1 pa rt bean) and placing it in an ultrasonication bath for 5 minutes. Compared to more traditional ag itation methods, sonication apparently makes LDopa more available to the solv ent by rupturing the cellular stru ctures in the bean. This method of L-Dopa extraction has not been researched for practical use. If this method were to be adapted for mediumto large-scale production of Mucuna, it would require investment in sonication water baths and safety equipment fo r the protection of the operators (e.g. sound barriers and silencers). Statement of Objectives Mucuna has great potential as a high-protein, high-starch food and feed for humans and monogastric livestock. However, the high levels of L-Dopa in this bean have limited its adoption for these purposes. Various methods of detoxifying Mucuna have been examined, but relatively little attention has been paid to the effects of methods on the nu tritional value. Some important experiments have validated certain aspects of Mucunas nutraceutical promise but additional research is required to validate many other ethno-pharmacological cl aims of its effectiveness. 48

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Therefore the aims of this di ssertation are to compare differe nt methods of detoxifying the Mucuna bean for monogastric consumption, to evaluate the nutritional value of the detoxified bean, and to examine effects of feeding the detoxified bean on the performance and health of monogastrics. An additional aim was to examine the anthelmintic potential of Mucuna. Specific objectives of the dissertation, examin ed in respective experiments, were as follows: Objective 1a: To determine the effect of ensi ling duration on the fermentation of Mucuna; Objective 1b: To study the effect of particle size of ensiled Mucuna on L-Dopa concentration, nutritive value, and fermentation characteristics; Objective 2: To determine the effect of sonication, or acid or alkaline solv ent extraction on the L-Dopa concentration and nutritive value of Mucuna beans; Objective 3: To determine the effect of feeding ens iled, acid extracted or alkali-extracted Mucuna bean on the performance, physiology and behavior of Sprague-Dawley rats; Objective 4: To determine if ingestion of Mucuna beans reduces helminth parasite infection in lambs. 49

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CHAPTER 3 EFFECT OF ENSILING ON L-DOPA AND NUTRITIONAL VALUE OF Mucuna pruriens Introduction Mucuna pruriens is a legume indigenous to Asia that grows in many tropical regions including Africa and the West Indies According to Ezeagu et al. (2003), Mucuna beans are high in protein (24-29%) and starch (39-41%). Adeb owale et al. (2005b) reported a higher protein range for Mucuna (33 to 38%), reflecting variation in growth environment. These nutritional attributes explain w hy various species of Mucuna are grown as a minor food crop in tropical countries despite their toxic properties. The ma jor toxic component of Mucuna compromising its usefulness as a food source for humans or livesto ck is its content of antinutritional compounds, particularly 3,4-dihydroxy-L-phenylalanine (L -Dopa), the chemical precursor to the neurotransmitter dopamine. Szabo and Tebbett (2 002) reported L-Dopa concentrations ranging from 4.47 to 5.39% L-Dopa in the bean, but wider ranges have been reported (3.1 to 6.7%; Daxenbichler et al., 1972). Although ruminants are not advers ely affected by ingestion of Mucuna (Burgos et al., 2002; Perez-Hernandez, 2003; Nyambati and Soll enberger, 2003; Castillo-Caamal, 2003ab; Eilitta et al., 2003; Matenga et al., 2003; Me ndoza-Castillo et al., 2003 ; Muinga et al., 2003; Chikagwa-Malunga et al., 2008b), nu merous publications report its toxic effects on monogastrics (Carew et al., 2002; Del Carmen et al., 2002; Flores et al., 2002). Most toxic effects in monogastrics arise directly from dopamines act ivity as a neurotransmitter involved in the regulation of the heart, vascular system, digestiv e tract, and excretory system, rather than from its well-known effect as a neurotransmitter in the brain (Szabo and Tebbett, 2002). Some consequences of increased peripheral dopamine in humans include orthostatic hypotension resulting in dizziness and in some cases stagge ring and increased heart rate, nausea, vomiting, 50

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and anorexia are also common side effects of ex cess L-Dopa ingestion, an d they are associated with the effects of peripherally produced dopamine on the dopamine receptors (Szabo and Tebbett, 2002). Safe levels of L-Dopa in monogastric livestock diets are considered to be 0.4% or less (Eilitta et al., 2003; Carew et al., 2003; Ferriera et al., 2003; Iyayi and Taiwo, 2003; Ukachukwu and Szabo, 2003). Studies indicate that proce ssing techniques can reduce the L-Dopa concentration of Mucuna beans to a safe level, enabling it to be us ed as a food source for monogastrics (Bressani, 2002). Processing methods that utilize heat decrease the L-Dopa concentration to or near a 1% level; permeation of the bean with hot water removes over 75% of the water-soluble L-Dopa, and boiling eliminates almost all (>99%) of the L-Dopa (Szabo a nd Tebbett, 2002). Extraction rates increase with increasing water temperature, allowi ng safe levels to be reached within 13 h at 40C, 3 h at 66C, and 40 min in boiling water (Teixeira and Rich, 2003). However, this method is not economically feasible for widespread use in developing countries (Gilbert, 2002) because heating fuel is expensive and copious amounts of water are required. Other treatment methods such as solvent extraction and ensiling are more practicable for de veloping countries. Since it is readily soluble in dilute acid so lutions, assays for determining th e concentration of L-Dopa in plants begin with hydrochloric aci d extraction. Acid-solvent extracti on can be just as effective as boiling water extraction (Myhrman, 2002), but this me thod depends on the availability of acids. Mucuna has been boiled and fermented to produce food products such as tempe (Egounlety, 2003), and mixtures of Mucuna and corn have also been ensiled (Matenga et al., 2003). In both instances the diges tibility of the bean increased after fermentation. According to Egounlety (2003), the crude protein (CP) concen tration was unaffected when tempe was made from Mucuna, but fermentation increased the protein dige stibility. Matenga et al. (2003) ensiled 51

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various mixtures of Mucuna and maize grain for 21 days and reported that the L-Dopa concentration was reduced by 10% for a 100% Mucuna sample and by 47% for a 30% Mucuna 70% maize mixture. The decrease in L-Dopa concentration due to Mucuna fermentation might be a useful strategy for making Mucuna safe for feeding to non-ruminants. However, the ensiling duration that is required for fermentation of Mucuna alone has not been determined and little is known about the nutritiv e value of fermented Mucuna. During ensiling, anaerobic microorganisms c onvert plant sugars into acids, thereby decreasing the pH. Quality silage is achieved when lactic acid is the predominant acid produced, as it is the strongest fermentation acid and it rapidly reduces th e pH, ensuring efficient nutrient preservation. When the initial concentrati on of water-soluble carbohydrates (WSC) exceeds 7%, a favorable homolactic fermentation usually occurs (Hong Yan Yang et al., 2006). Since Mucuna bean contains over 18% WSC, it is likely to be a good substrate for anaero bic microbial growth. Removal of L-Dopa from Mucuna beans depends on the particle size; smaller particles generally increase the surface area and promote the rate of interaction w ith extraction solvents (Teixeira et al., 2003). Larger particles, however can be obtained by cracking the bean by blunt force; this requires less prepara tion and less use of expensive equipment such as grinders. This study had two objectives. The first one was to de termine the effect of ensiling duration on the fermentation characteristics of Mucuna and the second to study the effect of particle size of ensiled Mucuna on L-Dopa concentration, nutritive valu e and fermentation characteristics. Materials and Methods Mucuna pruriens cv. Georgia bush, containing 25% CP 4.6% ether extract (EE), 17.3% neutral detergent fiber (NDF), 18.1% WSC, 38.2% starch, and 2.8% L-Dopa was obtained from the University of Georgia, Tifton, GA, USA. 52

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Effects of Ensiling Duration In the first of two experiments, beans were crushed in a roller mill (model 10004; Peerless International, Missouri, USA), collected in da rk plastic bags, mixed thoroughly and 1500 g subsamples were weighed into individual vacuum mini silo bags (26.5cm x 38.5cm; VacLoc Vacuum Packaging Rolls, FoodSaver, Neosho, MO, USA; Figure 3-1) in quadruplicate. To provide sufficient moisture for the fermentation, 900 ml of double-distilled water were added to the beans in each bag. A vacuum sealer (V 2220, FoodSaver, Neosho, MO, USA) was used to remove residual air from the bags Individual mini-silos were wr apped in brown paper bags and kept in a dark room at room temperature (18 to 25oC) for up to 28 days. The dark ensiling conditions were used to prevent the de gradation of light-sensitive L-Dopa. Figure 3-1. Method of ensiling Mucuna beans: A) vacuum sealing mi ni silos, B) weighing before and after ensiling, C) measuring th e pH; note excessive gas produced. The mini-silos were inspected daily and manually vented by pr icking with a pin to remove excessive gas accumulation when necessary (Fig ure 3-3). Pin holes we re immediately sealed with silo-tape after vent ing. Four bags containing Mucuna were opened after 0, 3, 7, 21, and 28 53

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days of ensiling. After ensiling, the contents of each bag were analyzed for dry matter (DM), pH, and concentrations of volatile fatty acids (VFA ), lactic acid, CP, and ammonia nitrogen (NH3-N). A pH of 4.6 or lower was taken to indicate adequate fermentation because this pH represents the typical minimum value for ensiled legumes (Heinr ichs and Ishler, 2000). Samples with a pH of 4.6 or lower were also tested for L-Dopa. Effects of Particle Size of Ensiled Mucuna Crushed Mucuna beans were sieved through a 6-mm (coarse) screen (USA Standard Testing Sieve, Fisher Scientific) or ground in a Wiley mill to pass through a 4-mm (medium) or a 2-mm (fine) screen (Arthur H. Thomas Compa ny, Philadelphia, PA, USA). Samples (1500 g) of each particle size were weighed into vacuum pl astic bags in quadruplicate. Double-distilled water (900 ml) was added to each bag and the bags were sealed and ensiled for 28 days based on the results of Experiment 1. Upon opening, mini-silos were subject to pH me asurement, then subsampled for analyses of DM, NDF, EE, gross energy (GE), L-Dopa, CP, and NH3-N, WSC, starch, mold and yeast counts, VFA, and aerobic stability (AS). Chemical Analysis Mucuna silage extract was obtained by blending 20 g of the ensiled bean with 200 ml of distilled water for 30 s at high speed in a blender (31BL91 Waring Commercial Blender, Dynamics Corporation of America, New Hartfo rd, Connecticut, USA). The mixture was filtered through two layers of cheesecloth and the pH measured (Accumet pH meter, model HP-71, Fischer Scientific, Pittsburg, PA, USA). The filtrate was centrifuged (1369 g for 20 min at 4oC) and the supernatant stored at -20oC for subsequent determination of VFA and NH3-N concentration. 54

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Mucuna silage samples were dispatched in a cooler (4oC) to the American Bacteriological & Chemical Research Corporation (Gainesville, Florida) for yeast and mold counts. Yeasts and molds were enumerated by pour plating with standard methods agar (SMA) to which 4% of chloramphenicol and chlortetracycline were added (Tournas et al., 1999). Dry matter concentration was determined after drying at 60oC for 72 hours and ash was measured by combustion in a muffle furnace at 550oC overnight. Dried samples were ground to pass through a 1-mm screen in a Wiley mill (Arthur H. Thomas Company, Philadelphia, PA, USA). Tota l N was determined by rapid combustion using a macro elemental N analyzer (Elementar, vari o MAX CN, Elementar Americas, Mount Laurel, NJ, USA) and used to compute CP (CP = N 6.25). The neutral detergent fiber (NDF) concentration was measured using the method of Van Soest et al. (1991) in an ANKOM 200 fiber analyzer (ANKOM Technolog ies, Macedon, NY, USA). Amylase was used in the analysis and the results were expressed on a DM basis. An adaptation of the Noel and Hambleton (1976) procedure that involved colorimetric quantification of N was used to determine NH3-N concentration with an ALPKEM auto analyz er (ALPKEM Corporati on, Clackamas, OR, USA). Volatile fatty acids were measured using the method of Muck and Dickerson (1988) and a high performance liquid chromatograph (Hitachi, FL 7485, Tokyo, Japan) coupled to a UV Detector (Spectroflow 757, ABI Analytical Kratos Division, Ra msey, NJ, USA) set at 210 nm. The column was a Bio-Rad Aminex HPX-87H (Bio-R ad laboratories, Hercules, CA, USA) with 0.015M H2SO4 mobile phase and a flow rate of 0.7 ml/min at 45C. To measure AS, thermocouple wires were placed at the center of a mini-silo bag containing 1000 g of silage and each bag was placed in an open-top polystyrene container covered with a brown paper bag to maintain da rk conditions while allowing adequate aeration. 55

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The thermocouple wires were connected to data loggers (Campbell Scientif ic Inc. North Logan, UT, USA) that recorded the temperature every 30 minutes for 657 hours. The time that elapsed prior to a 2C rise in silage temperature above ambient temperature was denoted AS. Ambient temperature was monitored every 30 minutes with three thermocouple wires. Concentration of LDopa was measured using the method of Siddhuraju and Becker (2001b) and a high performance liquid chromatography system (H ewlett Packard HP1100) and vari able wavelength UV detector set at 280 nm. The column used was an Apollo C18 (4.6 x 250 mm) column with a 19.5 ml methanol: 1 ml phosphoric acid: 975.5 ml water (pH 2; v/v/v) mobile phase flowing at 1 ml/min at 25C. Water-soluble carbohydrat es were quantified by the an throne method (Ministry of Agriculture, Fisheries and F ood, 1986). Starch was measured by a modification (Hall, 2001) of the glucose-oxidase-peroxidase (GOP) method of Holm et al. (1986). Ether extract was determined using the soxhlet procedure (Associ ation of Official Analytical Chemists, 1984). Gross energy (GE) was determined by an adiabatic bomb calorimeter (1261 isoperibol bomb calorimeter, Parr Instrument Company, Moline, Illinoi s, USA), using benzoic acid as a standard. Statistical Analysis Each experiment had a completely randomi zed design with 4 replicates. Data were analyzed with the MIXED procedure (SAS 9.1, SAS Inst. Inc., Cary, NC, USA). The following model was used to analyze the results: Yij = + Ti + Eij Where: Yij = dependent variable = general mean Ti = effect of the ith treatment Eij = experimental error When the treatment effect was significant ( P < 0.05), means were separated with a PDIFF statement. Tendencies were declared at P > 0.05 and 0.10. Orthogonal polynomial contrasts 56

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(linear, quadratic and cubic) were used to evalua te the effects of ensiling duration in Experiment 1 and increasing the particle si ze (2, 4, and 6 mm) in Experiment 2 on silage quality measures. Results Effects of Ensiling Duration A pH of 4.5 (Table 3-1) and an L-Dopa concentration of 1.3% (Figure 3-2) were obtained after 28 days of ensiling. During the fermentation, the pH decreased cubically, whereas concentrations of NH3-N, lactate, isobutyrate and isovalera te increased non-linearly. Butyrate was not detected in the silages. The NH3-N concentration remained below the threshold of 10% of total N throughout the ensiling period, lactate concentration had increased by 74% by day 28 and therefore lactate was the predominant fermen tation acid. The lactate:acetate ratio increased cubically from 1.12 at day 0 to 3.60 at day 28. Dr y matter losses were not detected in any treatment. Figure 3-2. The L-Dopa concentr ation of unensiled and ensiled Mucuna bean. Means without a common superscript letter differ ( P < 0.05); error bars denote standard error. 57

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Table 3-1. Fermentation characteris tics and L-Dopa concentration of Mucuna silage after various ensiling durations Days Item 0 3 7 21 28 SEMA Polynomial contrasts pH 6.2a 6.1a 5.4b 4.7c 4.5c 0.1 C NH3-N, % DM 0.19a 0.26a 0.29a 0.31a 0.39b 0.02 C NH3-N,% total N 4.9d 6.8c 8.1b 7.8bc 9.4a 0.4 C Lactate, % DM 0.66cd 0.29c 1.97bcd 2.06bc 2.57ab 0.57 C Acetate, % DM 0.73 0.43 0.61 0.53 0.85 0.17 NS Propionate, % DM 0.15 0.93 0.64 0.09 0.21 0.33 NS Iso-butyrate, % DM 0.47c 0.26c 0.32c 0.84b 1.35a 0.10 C Iso-valerate, % DM 0.23d 0.61c 0.95a 0.59c 0.64bc 0.08 C Total VFA, % DM 1.00 1.62 2.30 1.49 2.62 0.62 NS Lactate:acetate 1.12bc 0.65c 3.09abc 3.80ab 3.60abc 1.00 C A SEM = standard error of mean; C = cubic; NS = not significant; within a row, means without a common superscript letter differ ( P < 0.05). Effects of Particle Size of Ensiled Mucuna The L-Dopa concentration was reduced by 64, 42 and 57% in coarse, medium and fine particles, respectively (Figure 3-3). Particle size did not affect CP, starch, fat, or NDF concentrations or DM-losses (T able 3-2). The WSC concentrati on of ensiled coarse/medium and fine particles was reduced by 45 and 73%, respec tively. Gross energy values were unaffected by ensiling, but ensiled medium and fine particles ha d 6-7% less GE than ensiled coarse particles. The ash concentration of coarse and control particles were similar but ash concentration increased by more than 44% by en siling medium and fine particle s. Ensiling decreased the pH by 22-26%, but particle size did not affect this reducti on (Table 3-3). Ensilin g increased lactate, isobutyrate, total VFA, valerate, and NH3-N concentrations. Ensiling also increased acetate, propionate and isovalerate concentr ations of fine and coarse but not medium particles. Lactate was the main fermentation acid that was produced and the lactate:acetate ratio exceeded 3.0 in all ensiled treatments. Among ensiled samples, co arse particles had less lactate and more NH3-N 58

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than fine or medium particles, and a lower lactate to acetate ratio. Mold and yeast counts (Table 3-4) were unaffected by partic le size, and due to low numbers of these microbes, AS was maintained beyond 657 hours in all treatments. Table 3-2. Chemical composition of unensiled Mucuna (CON) and Mucuna ensiled at various particle sizes for 28 days Ensiled Item CON 2 mm 4 mm 6 mm SEM Polynomial contrasts DM, % 91.2a 37.6b 39.8b 38.9b 1.0 NS DM-loss, % NA 1.1 0.9 0.8 0.4 NS CP, % DM 25.0 23.2 23.7 24.4 0.4 NS Ash, % DM 6.0c 13.4a 10.7ab 8.8bc 1.0 L GE, cal/g 4055ab 3859b 3903b 4135a 71 NS Starch, % DM 38.2 38.0 39.6 38.4 1.0 NS WSC, % DM 18.1a 4.8c 10.1b 10.0b 1.2 L Fat, % DM 4.6 4.8 4.7 4.9 0.2 NS NDF, % DM 17.3 19.9 18.7 18.1 1.2 NS SEM = standard error of mean; L = linear effec t; NS = not significant; within a row, means without a common supers cript letter differ ( P < 0.05). Control2mm 4mm6mm 0 1 2 3 a b c cParticle size % ) a ( p L-Do Figure 3-3. Effect of particle size on L-Dopa concentration of ensiled Mucuna. Means without a common superscript letter differ ( P < 0.05); error bars denote the standard error. 59

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Table 3-3. Fermentation characteristics of unensiled Mucuna (CON) and Mucuna ensiled at various particle sizes for 28 days Ensiled Item CON 2 mm 4 mm 6 mm SEMA Polynomial contrasts pH 6.18a 4.58b 4.80b 4.73b 0.11 NS Lactate, % DM 0.66c 6.40a 6.44a 4.42b 0.56 NS Lactate, % total acids 42.33 53.66 57.57 46.23 13.62 NS Acetate, % DM 0.73b 1.28a 1.05ab 1.30a 0.15 Q Propionate, % DM 0.15b 0.46a 0.31ab 0.45a 0.09 NS Iso-butyrate, % DM 0.47b 3.01a 2.76a 2.61a 0.21 NS Butyrate, % DM 0.00b 0.47a 0.37ab 0.19ab 0.12 NS Iso-valerate, % DM 0.23b 0.88a 0.73ab 1.07a 0.17 NS Valerate, % DM 0.00b 0.01a 0.01a 0.01a 0.00 NS Total VFA, % DM 1.00b 5.50a 4.70a 5.07a 0.36 NS Lactate:acetate 1.12c 5.03ab 6.16a 3.41b 0.64 Q NH3-N,% DM 0.18c 0.51b 0.51b 0.56a 0.01 NS NH3-N,% total N 4.41c 11.56b 11.88b 13.03a 0.28 L A SEM = standard error of mean; L = linear effec t; Q = quadratic effect; NS = not significant; within a row, means without a common superscript letter differ ( P < 0.05). Table 3-4. Microbial counts and aer obic stability (AS) of unensiled Mucuna (CON) and Mucuna ensiled at various particle sizes for 28 days Ensiled Item CON 2 mm 4 mm 6 mm SEM Yeasts, log cfu/gA <1.0 3.0 <1.0 <1.0 N/A Molds, log cfu/gA <1.0 2.8 2.5 3.0 0.3 Aerobic stability, hours N/A >657 >657 >657 N/A A cfu/g = Colony-forming units per g of Mucuna silage; within a row, means without a common superscript letter differ ( P < 0.05). Discussion Characteristics of well preserved si lages include a pH below 4.0, an NH3-N concentration below 10% of total N, and a lactate:acetate ratio above 2.0 (Owens et al., 1999). Legume silages tend to have a pH range of 4.6 to 5.2 (Heinrichs a nd Ishler, 2000). Therefore, it took 28 days to achieve the minimum typical pH of legume silages. The NH3-N concentration in Experiment 1 increased to 9.4% of total N after 28 days of ensiling, indicating that proteolysis was not 60

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excessive. The predominance of lactate in the tota l acid concentration indicates that a homolactic fermentation occurred and reflects the utilizati on of WSC by lactic acid bacteria. This lactate accumulation led to the 27% drop in pH to a valu e of 4.5 after 28 days of fermentation. Since legume silages typically do not achieve a lowe r pH, this ensiling duration was used in Experiment 2. Ensiling for 28 days resulted in a 54% decline in L-Dopa concentration to 1.2% of DM. This was higher than the gene rally accepted safety threshold (< 0.4%) for consumption of Mucuna by monogastric livestock (Eilitta et al., 2 003; Carew et al., 2003; Ferriera et al., 2003; Iyayi and Taiwo, 2003; Ukachukwu and Szabo, 2003). In Experiment 2, concentrations of NH3-N were slightly above the threshold of 10% indicating that minimal prot eolysis occurred (Seglar, 2003). The relatively high lactate concentrations and lactate:acetate ratios beyond the critic al value of 2.0 in all ensiled treatments indicate the predominance of efficient hom o-fermentative pathways during ensiling of Mucuna. Ensiling decreased the L-Dopa concentration by 61% relative to the control, exceeding the 10 to 47% decrease reported by Matenga et al (2003) after ensiling different mixtures of Mucuna with maize grain for 21 days. The 10% re duction in L-Dopa content occurred in a 100% Mucuna with 0% maize mixture, and th e 47% reduction occurred for a 30% Mucuna with 70% maize mixture. More L-Dopa was lost as more maize was included in the mixture probably because more fermentable sugars that enhanced the fermentation were supplied as the proportion of maize increased. The r eason why fermentation of Mucuna was more efficient at L-Dopa extraction in this study than in th e previous one is probably attribut able to differences in soluble carbohydrate concentrations of Mucuna, as well as the longer ensi ling duration in this study. Ensiling for 28 days reduced L-Dopa concentrations to about 1%, which was higher than the threshold of 0.4% considered safe for c onsumption by monogastric livestock. This ensiled 61

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Mucuna should be fed with other i ngredients in diets of monogast rics to dilute the L-Dopa ingested and avoid excessive L-Dopa intake. Particle size affected L-Dopa concentrati on of ensiled beans in a quadratic manner. Inexplicably, the medium sized particles had higher residual levels of L-D opa relative to the fine and coarse particles. Nevertheless, L-Dopa concentration was markedly reduced by ensiling without affecting CP concentration at each partic le size. Apart from that of Matenga et al. (2003), no published studies were found on the effects of ensiling Mucuna alone on its L-Dopa concentration and nutritional value. However, Egounlety (2003) also reported that the CP content of Mucuna tempe did not change after several boi ling steps and a fermentation process that collectively reduced the L-Dopa content and increased proteolysis. Greater ammonia-N concentrations in ensiled versus unensiled Mucuna in thus study, also indicate that ensiling increased proteolysis. The fact that ensiling produced normal ferm entation characteristics and decreased the LDopa concentration without advers ely affecting concentrations of most nutrients indicates that Mucuna silage can be of value as a food and feed component in the diet of monogastrics. However, care should be taken to ensure that total L-Dopa intake does not exceed levels associated with side effects. In humans, a daily L-Dopa intake of 0.25 g/day is used as a starting dose for Parkinsons patients in order to mini mize side effects (Szabo and Tebbett, 2002). The body progressively adjusts to inge sted L-Dopa, therefore this st arting dose could be increased by 0.5 g every 7-10 days up to a maximum th erapeutic dose of 8 g L-Dopa/day. Relative to other detoxification methods, such as solvent extraction at variable pH (Diallo et al., 2002 and Teixeira et al., 2003) and therma l processing (Wanjekeche et al., 2003), ensiling is a simpler, relatively inexpensive procedure th at does not require purchasing heating fuel, acid 62

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or alkaline solvents, or using copious amounts of water. Furthermore, unlike solvent extraction, ensiling does not lower the CP content or make the bean much darker due to formation of melanin (Teixeira et al., 2003; Wa njekeche et al., 2003). However, ensiling for 28 days did not reduce the L-Dopa concentration as much as th ese more conventional detoxification methods. Future research should investig ate if longer ensiling durations produce greater reductions in the L-Dopa concentration. Coarse particles of Mucuna ensiled well and resulted in as much L-Dopa removal as fine particles. This implies that for ensiling, beans can be crushed to an approximate particle size of 6 mm and this can easily be achieved by applying a blunt force to crack the beans into a few pieces. Therefore, no mechanical grinders are required and this represents an additional advantage over the solvent extraction method wher e a fine particle size must be achieved for successful detoxification. Conclusion In the current study, the pH of ensiled Mucuna was reduced to 4.5 within 28 days, which is the typical minimum pH value for legume silages. This ensiling duration decreased the L-Dopa concentration from 2.8 to 1.3% (Experiment 1) or 2.8 to 1.2% (Experiment 2). The lactate:acetate ratio of the en siled bean was high because lact ate dominated the fermentation. Except for decreasing the WSC c oncentration, the chemical con centration of the bean was preserved during ensiling. Mold and yeast counts were low and AS was maintained for over 657 hours. Based on the fermentation characterist ics, good nutritional comp osition, extensive AS, and reduction of L-Dopa concentration, ensiling is a promising method of processing Mucuna beans for monogastric consumption. The aerobic stability of beyond 657 hours indicates that the ensiled bean can be stored for long periods at room temperature. Coarse particles of Mucuna ensiled well and resulted in as much L-Dopa re moval as fine particles, and had higher energy 63

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and WSC concentrations. Therefore, mechanical or electrical grinders are not required for processing beans to be ensiled; application of a blunt force that will split the beans into a few pieces is sufficient for ensiling. Ensiled Mucuna should be fed with other dietary ingredients to moderate L-Dopa ingestion by m onogastrics because ensiling doe s not completely reduce the LDopa concentration to safe levels. Future rese arch should investigate if longer ensiling durations (>28 days) produce greater reductions in the L-Dopa concentration. 64

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CHAPTER 4 EFFECT OF SONICATION AND SOLV ENT EXTRACTION ON L-DOPA AND NUTRITIONAL VALUE OF Mucuna pruriens Introduction Malnutrition in developing countries is due in part to insufficient access to affordable protein sources. Diets of many children in such c ountries lack protein and instead consist mainly of cereal-based porridge that is bulky, low in energy and nutrients, and high in antinutrient concentration (Adebow ale et al., 2005a). Mucuna pruriens, a legume indigenous to tropical regions, can be used to incr ease the dietary protein for such children. The beans of Mucuna pruriens are high in nutrients including protein ( 25-38%), starch (39-41 %), and fiber (4%; Ezeagu et al., 2003; Adebowale et al., 2005b). Ade bowale et al. (2007) co mpared the amino acid profile of Mucuna with human protein requirements suggested by the Food and Agriculture Organization (FAO), World Heal th Organization (WHO), and th e United Nations (UN/ONU) and reported that the bioavailability and amino acid concentrations of Mucuna protein isolates exceeded recommended levels for all but methionine and cysteine. The lysine concentration of Mucuna is particularly high (B ressani, 2002), therefore Mucuna is a valuable supplementary protein source to cereal-based diets which are known to be lysine deficient. The chemical composition of the beans varies with cultivar, geographical location, maturity at harvest, and bean color (St-Laurent et al., 2002; Ezeagu et al., 2003). Mucuna contains anti-nutritive factors (ANF) a nd the most potent and problematic ANF in the Mucuna bean is L-Dopa (Ukachukwu et al., 2002), the concentration of which ranges from 3 to 7% on a dry basis (Daxenbich ler et al., 1972). Symptoms of Mucuna intake in humans and monogastric livestock include reduced feed inta ke, weight loss, diarrhea, vomiting, and skin lesions (Del Carmen et al ., 2002; Flores et al., 2002; Szabo and Tebbett, 2002). 65

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Studies indicate that some pr ocessing techniques can reduce Mucunas L-Dopa concentration to the safe threshold of < 0.4% L-Dopa (Eilitta et al ., 2003). L-Dopa is readily soluble in dilute solutions of hydrochloric aci d and traditional assay techniques for determining the level of L-Dopa in a sample begin by pe rforming a total extracti on in hydrochloric acid (Daxenbichler et al., 1972). Acidif ication of water to pH 3 allows extraction of the L-Dopa in Mucuna beans at 1-mm particle size to safe levels in less than 8 hours (Teixeira et al., 2003). However, this treatment could result in protein lo ss because of the increased protein solubility at pH less than the isoelect ric point (pH 4.0-5.0) of Mucuna protein (Adebowale et al., 2007). Alkaline conditions may al so facilitate the inacti vation of L-Dopa in Mucuna beans. Although limited information has been published on solubility of L-Dopa in alkaline solutions, Diallo et al. (2002) reported th at a calcium hydroxide solution was more effective than water for removing L-Dopa from Mucuna bean. Soaking the beans in 4% calcium hydroxide solution for 48 hours reduced the L-Dopa concentration to 0.001% Teixeira et al. (2003) also reported that extraction of Mucuna beans (1 mm particle size) in NaOH solution at pH 11 reduced L-Dopa to safe levels (<0.4%) in less than 8 hours. However, Teixeira et al. (2003) and Wanjekeche et al. (2003) reported that melanin is produced when Mucuna L-Dopa is extracted at alkaline pH and this makes the beans black. Melanin has been an ecdotally associated with the formation of melanoma in some studies (Dollery, 1999; Lete llier et al., 1999; Si ple et al., 2000), but no evidence for this association was found in othe r studies (Weiner et al ., 1993; Pfutzner and Przybilla, 1997; Fiala et al., 2002) Nevertheless, the black color of the acid or alkali-extracted bean may reduce its acceptability. According to Wa njekeche, beans cooked in acid solutions are a lighter shade of black than beans cooked in alka line solution, therefore th ey may be viewed as 66

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more acceptable. Effective L-Dopa detoxification methods that dont adversely affect the color or nutritional value of the bean are needed. Sonication is a method used in more recent la boratory L-Dopa extraction procedures (St. Laurent et al., 2000) and it is not associated with discol oration of the beans. St-Laurent et al. (2002) reported 5 minutes to be the most effec tive duration for sonication. However, effects of sonication on the nutritive value of Mucuna are unknown. Successful removal of L-Dopa from Mucuna beans with solvents depends on the particle size; smaller particles generally increase the surface area and the solid-liquid interaction, promoting the rate of L-Dopa transfer (Teixeira et al., 2003). In contrast, larger particles require less preparation and less expensive equipment such as grinders. The objective of this study was to examine the effects of met hod of extraction of finely (1 mm) or coarsely (6 mm) ground Mucuna beans on the L-Dopa content and nutritional composition. Methods examined included extraction in either acetic acid (pH 3) or sodium hydroxide (p H 11) for 8 hours or extraction by sonication (SON) in water (pH 7) for 5 minutes. Materials and Methods Extraction Methods Mucuna pruriens cv. Georgia bush was obtained from the University of Georgia, Tifton, GA, USA. Mucuna beans were crushed (Roller Mill model 10004, Peerless International, Missouri, USA) and either sieved to pass through a 6-mm screen (USA Standard Testing Sieve, Fisher Scientific, Pittsburgh, PA, USA) or groun d in a Wiley mill to pass through a 1-mm screen (Arthur H. Thomas Company, Philadelphia, PA, USA). Twenty-four representative 50-g samples (8 per treatment) of fine (1 mm) or coarse (6 mm) particles were subjected to sonication (SON) in water (neutral pH) or soaked in acidic (A CD) or alkaline (BAS) solutions. The ACD solution was brought to pH 3 by diluting 0.8 ml of a 25% (v /v) acetic acid solution in 2 L of distilled 67

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water. The alkaline solution was brought to pH 11 by dissolving 0.1 g of sodium hydroxide in 2 L of distilled water. Each solution was shaken (Eberbach shaker, Michigan, USA) at room temperature for 8 hours (Figure 42), then sieved through four layers of cheesecloth and a Whatman #1 filter paper (1001-240, Fisher Scien tific, Pittsburgh, PA, USA). The residue was subsequently rinsed with 1 liter of distilled-de ionized water. Samples we re also submerged in 2 L of water (pH 7.3) within a sonication bath (Branson Ultrasonics, Conne cticut) and sonicated for 5 minutes at room temperature. For each treatment, pairs of replicate residues were composited to provide sufficient sample for chemical analysis (n=4). Chemical Analysis Sonicated and solvent-extracte d residues were dried at 55 oC to 97% DM prior to further analysis. Dried samples were ground to pass thro ugh a 1-mm screen in a Wiley mill (Arthur H. Thomas Company, Philadelphia, PA). Dry matter concentration was determ ined after drying at 60oC for 72 hours and ash was measured by combustion in a muffle furnace at 550oC overnight. Gross energy levels were determined by an adiabatic bomb calorimeter (1261 isoperibol bomb calorimeter, Parr Instrument Company, Moline, Illi nois, USA), using benzoic acid as a standard. Total N was determined by rapid combustion using a macro elemental N analyzer (Elementar, vario MAX CN, Elementar Americas, Mount Laurel, NJ) and used to compute CP (CP = N 6.25). The neutral detergent fiber ( NDF) concentration was measured using the method of Van Soest et al. (1991) in an ANKOM 200 Fiber Analyzer (ANKOM Technologies, Macedon, NY). Amylase was used in the analysis and the results were ex pressed on a DM basis. The anthrone method (Ministry of Agriculture, Fisheries and Food, 1986) was used to quantify water-soluble carbohydrate (WSC). Starch was m easured by a modification (Hall, 2001) of the 68

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glucose-oxidase-peroxidase met hod of Holm et al. (1986). Ether extract (EE) was determined using the soxhlet procedure (Association of Official Analytical Chemists, 1984). The L-Dopa concentration of the Mucuna beans was measured using the method of Siddhuraju and Becker (2001b) a nd a high-performance liquid ch romatography system (Hewlett Packard HP1100) with an autosampler, degasser binary pump modules, a nd variable wavelength UV detector set at 280 nm. The column used was an Apollo C18 (4.6 x 250 mm) column with a 19.5 ml methanol: 1 ml phosphoric acid: 975.5 ml wate r (pH 2; v/v/v) mobile phase flowing at 1 ml/min at 25C. Statistical Analysis The experiment had a completely randomized design involving 7 treatments: untreated control, and acid, alkali, or soni cation treatments of 1and 6-mm beans. Each treatment had 4 replicates (n=4) and all values reported are least squares mean s. The following model was used to analyze the results: Yij = + Ti + Eij Where: Yij = dependent variable = general mean Ti = treatment effect (fixed effect representing Mucuna processing method and particle size) Eij = experimental error Data were analyzed with the MIXED pro cedure (SAS 9.1, SAS Inst. Inc., Cary, NC, USA). Significance was declared at P < 0.05 and means were separated with a PDIFF statement. Tendencies were declared at P > 0.05 and 0.10. Results All processing methods reduced L-Dopa concentrations of fine Mucuna particles from 2.8% to less than 0.2% (Figure 4-1) Acid and alkali treatments made the solvents and extracted bean residues darker (Figure 4-2). 69

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Figure 4-1. The L-Dopa concentration of fine (1 mm) or coarse (6 mm) Mucuna particles subjected to acid extraction (ACD), alkali extraction (A LK), or sonication (SON). Means without common letters differ ( P < 0.05); error bars denote standard error. All methods also reduced CP and WSC of fine particles by 24-31% and 78-81%, respectively (Table 4-1) and increased their NDF and starch concentrations by at least 62 and 14%, respectively. Fat concentration of fine part icles was reduced from 5.5% to 4.2% by SON, whereas ACD and ALK reduced their GE values by approximately 10%. The ash concentration of fine particles was increased by 88% and 35% by ALK and SON, respectively. Sonication and ACD did not reduce L-Dopa concentration of co arsely ground beans but ALK reduced it from 2.8% to 2%. Sonication reduced CP, WSC, and fat concentration of coarse particles by 6, 17, and 27%, respectively. The ALK treatment increased their starch concentration by 17% but decreased their WSC concentration by 78% The ACD treatment increased the NDF concentration of coarse partic les by 35% but decreased their WS C and fat concentrations by 51% and 31%. Ash concentration and GE of coarse part icles were not affected by any of the treatments. 70

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Table 4-1. Effect of processing method on the chem ical composition of fine (1 mm) and coarse (6 mm) Mucuna beans Item Unground ControlA ACDB 1 mm ALKC 1 mm SOND 1 mm ACDB 6 mm ALKC 6 mm SOND 6 mm SEM Dry matter, % 95.4 94.9 95.3 95.6 95.1 95.5 95.1 0.2 Crude protein, % DM 25.4a 19.3c 17.9cd 17.4d 25.3ab 24.6ab 23.9b 0.5 Ash, % DM 6.0c 7.9bc 11.3a 8.1b 6.6bc 6.9bc 7.3bc 0.7 Gross energy, Kcal/g 4.1a 3.6b 3.6b 3.8ab 4.0ab 3.9ab 3.9ab 0.13 Starch, % DM 38.2b 45.9a 46.2a 43.7a 36.8b 44.8a 34.6b 1.5 WSC, % DM 18.1 a 3.8 d 3.9 d 3.5 d 8.8 c 13.3b 15.0b 0.8 Fat, % DM 5.5a 5.9a 5.6a 4.2b 3.8c 3.6c 4.0b 0.4 NDF, % DM 17.3 e 32.0 b 38.0a 28.1bc 23.4c 21.0de 20.3de 1.9 Within a row, means without a common superscript letter differ ( P < 0.05); A untreated beans; B acid-treated beans; C alkali-treated beans; D sonicated beans; WSC = water-soluble carbohydrate; NDF = neutral detergent fiber. Discussion Successful removal of L-Dopa from Mucuna beans with solvents depends on the particle size because smaller particles in crease the surface area and the solid-liquid interaction, which promotes the rate of transfer of solute at the particle surface (Teixeira et al., 2003). Safe L-Dopa levels in Mucuna beans destined for monogastric livestock consumption are considered to be 0.4% or less (Eilitta et al., 2003). All extraction methods were equally effective in reducing the L-Dopa content of fine Mucuna particles to < 0.2% and thus making them safe for consumption by monogastrics. However, the L-Dopa content of coarse Mucuna particles was not decreased by ACD and SON and the 29% reduction by ALK treat ment was far less than that of the ALK treatment of fine particles (100%). These particle-size dependent responses are in agreement with Teixeira et al. (2003), who showed that LDopa removal at pH 3 or 11 depended on particle size was more effective in beans ground to a 1-mm particle size versus th at ground to 2-, 4-, and 8-mm sizes. This is because larger particles ha ve less surface area, and therefore result in less efficient extraction of L-Dopa. The efficacy of the acid and alkali-treatment of fine particles also agrees with the observations of Teixeira et al. (2003). 71

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The CP concentration of fine particles was reduced by 24-31% in all treatments but that of coarse particles was only reduced by SON. The latter was likely because of the cell rupturing effect of sonication which would have exposed more of the protein to the solvent and thus increased solvent penetration. Mo st (67.5%) of the protein in Mucuna is water-soluble (Adebowale et al., 2007). Greater CP and WSC losses in finer particles were because of the greater surface area exposed to the solvents. This is in agreement with Myhrman (2002) and Teixeira et al. (2003) who reported CP losse s of 11% and up to 50%, respectively due to leaching after soaking of finely ground bean samp les in acid or alkaline solutions. Losses of CP from ACD and ALK-treated fine particles we re also facilitated by the solubility of Mucuna protein. High protein solubility in water and alkaline pH conditi ons can increase protein losses in solutions, disrupt protein structur e and lead to degradation of certain amino acids (Adebowale et al., 2007). Water-soluble albumin is the dominant protein in Mucuna bean and Mucuna protein has its highest solubility at al kaline pH (8-12) with a maximum value of 96% at pH 12, whereas solubility at acidic pH (2-6) is less and minimum protein solubility in most solutions is at pH 4.05.0, which corresponds to the isoelectric pH of Mucuna protein (Adebowale et al., 2003; 2005a). For unknown reasons, the al kaline extraction of Mucuna did not lead to greater CP losses than the acidic extraction. More work should be don e on effects of these extraction methods on the true protein concentration of Mucuna. The increased starch content of fine particle s due to ACD, BAS or SON treatments of fine particles agrees with responses to acid or alkali extraction of Mucuna reported by Siddhuraju and Becker (2005). The increased starch content wa s due to partial loss of soluble components including WSC, protein, and L-Dopa, all of which decreased with solvent extraction relative to CON. The reduction in concentrat ion of these component s and the energy value of the bean, and 72

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the concomitant increases in starch and fiber concentration imply that solvent extraction and sonication modified the nutritive va lue of the bean and resulted in losses of key components. Figure 4-2. Detoxification of Mucuna bean through acid or alkali so lvent extraction: A) shaking for 8 hours, B) color changes resulting from solvent pH, and C) washing and filtering the detoxified beans. Although methionine and cysteine are limiting amino acids in the Mucuna bean, a key nutritional attribute is its high lysine concentration and bioava ilability (Adebowale et al., 2007). Cereal grains are typically used to alleviate hunger in developing countries but they are known to be deficient in lysine and thus not very eff ective in reducing malnutrition (Adebowale et al., 2005b). The lysine and am ino acid composition of Mucuna reportedly exceeds the recommended FAO/WHO/UN reference values for human diets, indicating that it is an excellent source of essential amino acids, and can be used to for tify cereal-based foods deficient in lysine (FAO/WHO/ONU, 1985). Further research should determine effects of the processing methods employed in this experiment on concentratio ns of lysine and other amino acids in Mucuna. 73

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The pH of the solvent is an im portant factor that can affect the success of L-Dopa removal from Mucuna and the residual nutritional quality. Severa l authors mention that due to formation of melanin, Mucuna beans are darker after acid or alka li extraction (Teixe ira et al., 2003; Wanjekeche et al., 2003). The darker color in the acid and alkali extracts was in agreement with such observations (Figure 4-3). Melanin is a metabolite of L-D opa characterized by its dark color. The conversion of L-Dopa into melanin re quires specific conditions and is most evident at alkaline pH (Teixeira et al., 2003; Wanjekeche et al., 2003); for example if bicarbonate is added to the cooking or so aking water of the Mucuna bean (Eilitta et al., 2003). Figure 4-3. Color changes after detoxification of Mucuna bean through A) Alkaline extraction at 1 mm particle size, B) Alkaline extraction at 6 mm, C) Acid ex traction at 1 mm, D) Acid extraction at 6 mm particle size. Diallo et al. (2002) successfully reduced the L-Dopa concentra tion to 0.001% after 48 hours of soaking cracked Mucuna beans in calcium hydroxide solu tion, but noted the remarkably dark coloration upon treatment. B eans cooked in acid solutions are lighter in color than beans 74

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cooked in alkaline solutions (Wanjekeche et al., 2 003). Adebowale et al. ( 2007) noted that darker colors occurred in sodium hydroxide solutions of pH 11 relative to less alkaline solutions. This causes concern because the effects of melanin on health are controversial. Melanin has been anecdotally associated with the formation of me lanoma in some studies (Dollery, 1999; Letellier et al., 1999; Siple et al., 2000), but no evidence for this association was found in other studies (Weiner et al., 1993; Pfutzner a nd Przybilla, 1997; Fiala et al., 2002). Discarding the solvent residue as in the current study, may reduce this concern. Neverthe less, the darker color of the extracted bean indicates the need for further investigation of concentratio ns of melanin residues in the detoxified bean. Conclusion The efficiency of L-Dopa removal in the solv ent extracts was mainly affected by particle size. Both acidic and alkaline solvents performed equally well at detoxifying fine particles of Mucuna bean to safe levels (<0.4% L-Dopa) but also reduced their WSC and CP concentrations and increased their starch and NDF concentrations However, these methods were not effective at detoxifying coarse Mucuna particles and they had less consis tent effects on their nutritive value. Effective detoxification of fine part icles occurred at the e xpense of CP and WSC concentration. Acidic and alkaline solvent extraction darkened the bean, suggesting that they increased the formation of melanin, a metabolite of L-Dopa characterized by its dark color. Future research should determine melanin concentr ations in acidor alka li-extracted beans as well as their concentrations of true protein and amino acids. Sonication prevented discoloration of Mucuna due to acid or alkaline extraction and it was also an effective method of detoxifyi ng fine but not coarse particles of Mucuna to safe levels (<0.4% L-Dopa). Sonic waves rupture cells, there by increasing solvent pene tration and particle dispersal while promoting the accessibility of th e solvent to L-Dopa. Th e limited surface area of 75

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the coarse (6 mm) particles combined with the la ck of penetration of th e sonic waves through the larger particles were likely re sponsible for the limited effec tiveness of sonication on coarse particles. Sonication generally resulted in similar modifications to the nutr itive value of the bean as acid or alkali solvent extraction but caused great er losses of fat from fine particles. 76

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CHAPTER 5 BEHAVIORAL, PERFORMANCE, AND PHYSIOLOGICAL RESPONSES OF RATS FED DETOXIFIED Mucuna pruriens Introduction The major problem that has compromised the usefulness of Mucuna pruriens as a food source is its concentration of antinutrients According to Szabo and Tebbett (2002) and Ukachukwu et al. (2002), 3,4-dihydr oxy-L-phenylalanine (L-Dopa) is the most potent toxic compound in the Mucuna bean, which contains between 3.1 and 6.7% L-Dopa (Daxenbichler et al., 1972). Szabo and Tebbett (2002) described the follo wing pharmacokinetic profile of L-Dopa, which illustrates the danger of introducing undetoxified Mucuna into the human diet. Approximately 33% of an orally administered dose of L-Dopa is absorbed from the gastrointestinal tract, primarily from the je junum in the small intestine. Peak plasma concentrations occur within 1 to 3 hours. Most of the successfully absorb ed L-Dopa is converted to dopamine in the periphery via action of th e enzyme L-aromatic amino acid decarboxylase (LAAD). Less than 1% of the administered dose enters the brain where it is converted to dopamine in the basal ganglia. In addition to dopamine, peripheral LDopa is also metabolized to melanin, norepinephrine, 3-methoxytyramine, methyldopa, 3,4-dihydroxyphenylacetic acid, and 3-methoxy-4-hydroxyphenylacetic acid (homovanillic acid). These metabolites are rapidly excreted in the urine such that approximately 80% of an administered dose is excreted within 24 hours, less than 1% of which remains unchange d. Consequences of increased peripheral dopamine in humans can include nausea, vomiting, and anorexia, orthostatic hypotension resulting in dizziness, staggering, and increased heart rate. Psychi atric disturbances have also been reported in patients receiving high doses of L-Dopa in a dose-dependent manner (Szabo and 77

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Tebbett, 2002). These can include nervousness, anxiety and agitation, in somnia, vivid dreams, confusion, delirium, depression and psyc hotic reactions with hallucinations. Cotzias et al. (1974) reported th at feeding rodents high levels of L-Dopa affected behavior; they expressed motor hyperactivity, muscle jerk s, stereotyped moveme nts, jumping, gnawing, corkscrew tails, ataxia, salivation, piloerection, re d muzzle, and convulsions. Most side effects arise directly from dopamines activity as a ne urotransmitter involved in the regulation of the heart, vascular system, digestive tract, and excretory system, rather than from its well-known effect on receptors in the brain (S zabo and Tebbett, 2002). Symptoms of Mucuna intake in broilers and pigs include weight loss, reduced feed intake, an d feed conversion efficiency (Flores et al., 2002; Del Carm en et al., 2002). Despite the health hazards caused by L-Dopa, the Mucuna beans high protein concentration makes it an important part of the diet in Asia, Africa, and South/mid-America. According to Ezeagu et al. (2003), Mucuna beans are not only high in protein (25-30%), but also in starch (39-41%). Adebowale et al. (2007) show ed that except for methionine and cysteine, concentrations of bioavailable amino acids in Mucuna protein isolates exceeded the values for human diets recommended by the Food and Agri cultural Organization (FAO), World Health Organization (WHO), United Nations (UN/ONU). Mucunas high lysine concentration makes it a suitable supplementary protein to cereal-based diets which are known to be lysine deficient (Adebowale, 2007). Mucuna could thus be used to alleviate malnutrition in developing countries, provided its L-Dopa concentrati on is effectively reduced (Bressani, 2002; Teixeira et al., 2003). The safety threshold is a bean L-Dopa concen tration of less than 0.4% (Eilitta et al., 2003; Carew et al., 2003; Ferriera et al., 2003; Iyayi and Taiwo, 2003; Ukachukwu and Szabo, 2003). Processing techniques have been evaluated that reduce the Mucuna L-Dopa concentration to safe 78

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levels (Bressani, 2002), but these techniques are often costly as th ey require expensive fuel to generate heat, copious amounts of water that is not always readily available, and they are labor intensive and time consuming. Few studies have examined the residual nutritional value of detoxified Mucuna bean and the effects of feeding it to monogastrics. A few promising detoxification methods were identified in preliminary studies. Ensiling the Mucuna bean for 28 days reduced the L-Dopa c oncentration by 54%, preserved the starch and protein concentrations, and re sulted in a product that had not deteriorated after 657 h of storage (Chapter 3). So lvent extractions for 8 hours at pH 3 or 11 almost eliminated (> 90% removal) Mucuna L-Dopa but also reduced the protein concentration by 24-31% (Chapter 4). The purpose of this study was to evalua te the effect of feeding detoxified Mucuna beans on performance, physiology and beha vior of Sprague-Dawley rats. Materials and Methods Mucuna Detoxification Mucuna pruriens cv. Georgia bush, were obtained fr om Dr. Sharad Phatak at the University of Georgia, Tifton, GA, USA. The th ree detoxification methods evaluated consisted of acid extraction, alkaline extraction and ensiling. Detoxification through acid or alkali solven t extraction Mucuna beans were ground in a Wiley mill to pass through a 1-mm screen (Arthur H. Thomas Company, Philadelphia, PA, USA). An acidic solution was brought to pH 3 by diluting 0.8 ml of a 25% (v/v) acetic acid solution in 2 L of distilled water. The alkali solution was brought to pH 11 by dissolving 0.1 g of sodium hydr oxide in 2 L of disti lled water. Suspensions (25 g/l) of Mucuna in the acid and alkaline solutions were shaken (Eberbach shaker, Michigan, USA) at room temperature for 8 hours, then filt ered through four layers of cheesecloth and a 79

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Whatman #1 filter paper (1001-240, Fisher Scien tific, Pittsburgh, PA, USA). The residue was subsequently rinsed with one liter of distilled-deionized water and dried at 55oC to 97% DM. Detoxification through ensiling Mucuna beans were ground in a Wiley mill to pass through a 6-mm screen (Arthur H. Thomas Company, Philadelphia, PA, USA). Ground beans were weighed (1500 g) into individual vacuum bags (26.5 x 38.5 cm, VacLoc Vacuum Packaging Rolls, FoodSaver, Neosho, MO, USA) and 900 ml of double distilled water we re added to provide sufficient moisture for fermentation. A vacuum sealer (V2220, FoodSav er, Neosho, MO, USA) was used to remove residual air from the bags and to heat seal them Bags were placed in brown paper bags and kept in a dark room at room temperature (18 to 25oC) for 28 days. The bags were inspected daily and manually vented by pricking with a pin to remo ve excessive gas accumulation when necessary. Pin holes were immediately sealed with silo-tape after venting. After ensi ling, the concentrations of each bag were dried at 55oC to 97% DM. All procedures we re performed under conditions of limited lighting since L-Dopa is lig ht sensitive. Upon detoxificati on, representative samples were analyzed for L-Dopa and nutri tional value (Table 5-1). Table 5-1. Chemical composition of unde toxified (control) and detoxified Mucuna beans Control Detoxification methoda Item Ensiling Acid extraction Alkaline extraction Crude protein, % DM 25.0 23.2 19.3 18.1 Ash, % DM 6.0 13.4 7.9 11.3 Gross energy, Kcal/g 4.1 3.9 3.6 3.6 Starch, % DM 38.2 38.0 45.9 46.2 WSC, % DM 18.1 4.8 3.8 3.9 Fat, % DM 4.6 4.8 5.9 5.6 NDF, % DM 17.3 19.9 32.0 38.0 pH 6.2 4.5 3.0 11.0 L-Dopa, % DM 2.8 1.2 0.1 0.0 WSC = water-soluble carbohydrate; NDF = neutral detergent fiber; a Chapters 3 and 4. 80

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Analysis of L-Dopa The L-Dopa concentration of detoxified Mucuna beans was measured using the method of Siddhuraju and Becker (2001b) a nd a high performance liquid ch romatography system (Hewlett Packard HP1100) with a variable wavelength UV detector set at 280 nm. The column used was an Apollo C18 (4.6 x 250 mm) column with a 19.5 ml methanol: 1 ml phosphoric acid: 975.5 ml water (pH 2; v/v/v) mobile phase flowing at one ml/min at 25C. Nutritional Value Analysis Dried samples were ground to pass through a 1-mm screen in a Wiley mill (Arthur H. Thomas Company, Philadelphia, PA), and ash wa s measured by combustion in a muffle furnace at 550oC overnight. Total N was determined by ra pid combustion using a macro elemental N analyzer (Elementar, vario MAX CN, Elementa r Americas, Mount Laurel, NJ) and used to compute CP (CP = N 6.25). NDF concentration was measured using the method of Van Soest et al. (1991) in an ANKOM 200 Fiber Analyzer (ANKOM Technologies, Macedon, NY). Amylase was used in the analysis and the result s were expressed on a DM basis. The anthrone method as described by the Ministry of Agricu lture, Fisheries and Food (1986) was used to quantify water-soluble carbohydrate (WSC). Starch was measured by a modification (Hall, 2001) of the glucose-oxidase-peroxidase (GOP) method of Holm et al (1986). Ether extract (EE) was determined using the soxhlet procedure (Associ ation of Official Analytical Chemists, 1984). Gross energy levels were determined by an adiabatic bomb calorimeter (1261 isoperibol bomb calorimeter, Parr Instrument Company, Moline, Illinoi s, USA), using benzoic acid as a standard. Mucuna silage extract was obtained by blending 20 g of the ensiled bean with 200 ml of distilled water for 30 s at high speed in a blender ( 31BL91 Waring Commercial Blender, Dynamics Corporation of America, New Hartford, Conn ecticut, USA). The mixture was filtered through 81

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two layers of cheesecloth and the pH measur ed (Accumet pH meter, model HP-71, Fischer Scientific, Pittsburg, PA, USA). Dietary Treatments The diets for each of the five treatments we re prepared by Harlan Teklad (Madison, WI, USA) and each consisted of 1.2 cm pellets and contained 25-26% CP, 9% ash, 37% carbohydrates, 4% fat and 2.7 Kcal/g GE. The treatments consisted of one control diet (CON) consisting of a commercial rat chow (8604 rodent diet Harlan Teklad, Madison, WI, USA) and four Mucunabased diets in which 10% of the commercial rat chow was replaced with either untreated Mucuna (MUC), or Mucuna beans detoxified by acetic acid extraction (ACD), sodium hydroxide extraction (BAS), or ensiling (SIL). Animals and Measurements Sixty 6to 8-week-old male Sprague-Dawley rats with an initial body weight of 200 grams were purchased from Harlan (Indianapolis, IN, USA). Rodents are commonly used as a suitable animal model for monogastrics in food safety and toxicology studies. Rats were individuallyhoused (Figure 5-1) in 40 x 30 x 20 cm cages and randomly assigned to the five treatments (n=12). All animals were housed at 20 1oC in a 12-h light/dark cycle with an ad libitum tap water and food supply. Experiments were performed according to the policies and guidelines of the Institutional Animal Care and Use Committee of the University of Florida, Gainesville, USA. Open field behavior analysis Animal behavior was evaluated on day 3 (Week 1) and day 10 (Week 2) using the open field test. The open field consis ted of a round grey plastic arena measuring 70 cm in diameter surrounded by a grey plastic wall 34 cm high, lit with three 40W bulbs (Figure 5-1). The floor of the arena was divided into severa l concentric units by black painte d lines, dividing the arena into 19 fields. This method is used to evaluate rode nt behavior (Carlini et al., 1986). Open field 82

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activity helps determin e locomotion and motor activity as well as speed (Vogel, 2002). Each week, the 60 rats were placed one at a time in the center of the arena for 5 minutes. The open field test was videotaped using a high-re solution video camera WV-CP244 (Panasonic, Secaucus, NJ, USA). Video analysis was perfor med using TopScan, Top View Animal Behavior Analyzing System (version 1.00, Clever Sys In c., Preston, VA, USA) by an unbiased and treatment-blinded technician. Performance and physiological analysis Rats were handled in a laminar flow hood (Figure 5-1). Feed intake duri ng the first 10 days of the trial was calculated on a daily basis by wei ghing the remaining feed (orts) left over from what was offered on the previous day. Animals were also weighed daily during the first 12 days of the trial and growth records us ed to determine average daily gain and total weight gain. After 14 days the rats were necropsied and the heart, liver, kidneys, spleen, and gonads were weighed. Organ weights were normalized to reflect percent of body weight. Figure 5-1. Experimental conditions for rat feeding trial: A) Rats were handled under the laminar flow hood and B) housed in individual cag es; C) A Sprague-Dawley rat and the open field test set-up. 83

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Clinical pathology analysis At the end of the trial (day 14), blood wa s collected post-anesthesia through cardiac puncture and stored in serum and EDTA vacutain er tubes (Vacuette, Greiner Bio-One NA, Inc, Monroe, NC, USA) for testing in a clinical pathology la boratory that performed a chemistry panel (vet-20 chemistry profile for rode nts) and complete blood counts (CBC). Statistical analysis Statistical analysis was performed with Gr aphPad Prism (version 4.00, GraphPad Software Inc., San Diego, CA, USA) and one-way analysis of variance (ANOVA) followed by Student NewmanKeuls multiple comparison test. In all cases differences were considered significant if P < 0.05. Results Performance Table 5-3 shows that dietary treatments did not affect DM intake or weight gain. Total DM intake, average daily DM intake, and total DM intake as a per cent of body weight, average daily DM intake as a percent of body weight, total weight gain, and average daily weight gain were not different among treatments (P > 0.05). However, rats fed MUC had numerically ( P > 0.1) lower values than those fed other diets. Behavior Dietary treatments did not aff ect distance traveled or number of line crossings in the open field test during day 3 (Week 1) or day 10 (W eek 2; Figure 5-2). Nevertheless, rats fed Mucuna consistently had numerically ( P >0.05) lower line crossings and distance traveled compared to CON and this trend was most evident in rats fed MUC and BAS on day 10. A malfunction of the Top View analyzing system allowed analysis of only six animals per treatment during day 3 (n=6) and three animals per tr eatment on day 10 (n=3). 84

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Table 5-2. Effects of feedi ng unprocessed or detoxified Mucuna pruriens on DM intake and growth of rats CONa MUCb ACDc BASd SILe Feed intake, g/11d 228.1 + 6.3 212.4 + 5.8 224.6 + 3.4 230.8 + 7.2 223.4 + 6.2 Daily DM intake, g 20.7 + 0.6 19.3 + 0.5 20.4 + 0.3 21.0 + 0.7 20.3 + 0.6 Feed intake, % BW 86.1 + 2.1 81.9 + 1.4 84.6 + 1.1 85.6 + 2.2 82.1 + 2.0 Daily DM intake, % BW 8.6 + 0.2 8.2 + 0.1 8.5 + 0.1 8.6 + 0.2 8.2 + 0.2 Weight gain, g/10d 59.5 + 3.3 58.2 + 5.3 61.8 + 2.6 65.7 + 2.2 66.9 + 2.5 Daily weight gain, g 5.9 + 0.3 5.8 + 0.5 6.2 + 0.3 6.6 + 0.2 6.7 + 0.2 Mean + standard error; within a row, means without a comm on superscript letter differ ( P < 0.05); a control diet, standard rat chow without Mucuna; b untreated Mucuna diet; c Mucuna beans detoxified by acetic acid extraction; d Mucuna beans detoxified by sodi um hydroxide extraction; e Mucuna beans detoxified by ensiling; BW = body weight; DM = dry matter. Compared to CON, feeding Mucuna diets significantly ( P < 0.01 and P < 0.05) reduced total (day 3 and day 10) distance traveled and total number of lin e crossings in the open field test. The reduction was more statistically significant ( P < 0.01) in rats fed MUC versus those fed detoxified Mucuna diets ( P < 0.05). There was a nume rical indication ( P > 0.05) that the reduction was less severe in rats fed ACD and SIL. Physiology Necropsy revealed that in all treatments the heart, liver, kidney and testicular weights remained unchanged relative to CON (Table 5-3). Levels of red blood cells were not different among treatments either. Feeding MUC increased spleen weight (splenomegaly) and monocyte occurrence (monocytosis) relative to CON, but feed ing the detoxified beans did not (Figure 5-3). Concentrations of alkaline phosphatase were in creased by 11-17% due to feeding detoxified beans instead of MUC, but all Mucuna treatments resulted in similar alkaline phosphatase concentrations as CON. Blood phosphorus conc entration was decreased by feeding MUC relative to CON (9.78 vs 10.74 mg/dl) but it was si milar in rats fed CON and detoxified diets. 85

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Figure 5-2. Effect of feeding detoxified Mucuna pruriens on A) distance traveled on day 3; B) open field line crossings on day 3; C) distance trav eled on day 10; D) ope n field line crossings on day 10; E) total distance traveled on both days; and F) total line crossings on both days. Means without a common superscript letter differ ( P < 0.05 *, P < 0.01 **). CON = control diet; MUC = untreated Mucuna diet; ACD = Mucuna beans detoxified by acetic acid extraction; BAS = Mucuna beans detoxified by sodium hydroxide extraction; SIL = Mucuna beans detoxified by ensiling. E rror bars denote standard error. 86

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Table 5-3. Effects of feedi ng unprocessed or detoxified Mucuna pruriens on organ weights and concentrations of monocytes, alkaline phosphatase, and phosphorus in the blood CONA MUCB ACDC BASD SILE Heart, % BW 3.84 + 0.04 3.76 + 0.05 3.88 + 0.06 3.68 + 0.05 3.71 + 0.07 Liver, % BW 45.2 + 0.6 44.8 + 1.6 45.1 + 1.1 44.2 + 1.0 44.6 + 0.9 Kidney, % BW 7.7 + 0.4 7.6 + 0.3 7.6 + 0.2 7.2 + 0.1 7.6 + 0.2 Testicles, % BW 12.4 + 0.7 13.6 + 0.3 13.2 + 0.2 12.9 + 0.3 12.8 + 0.3 Spleen, % BW 2.48b + 0.05 2.79a + 0.08 2.67ab + 0.05 2.54b + 0.06 2.64ab + 0.06 Red blood cells, x106/ul 7.4 + 0.1 7.3 + 0.1 7.4 + 0.1 7.3 + 0.1 7.1 + 0.1 Mean + standard error; within a row, means without a comm on superscript letter differ ( P < 0.05); A control diet, standard rat chow; B untreated Mucuna diet; C Mucuna beans detoxified by acetic acid extraction; D Mucuna beans detoxified by sodi um hydroxide extraction; E Mucuna beans detoxified by ensiling. Discussion In the current study, the feed intake, growth, behavioral and physiological effects on organ weight, complete blood counts, a nd blood chemistry profile were de termined to be tter understand food safety aspects of detoxified Mucuna bean. At the 10% of DM inclusion rate, feeding Mucuna did not significantly affect rat behavior in the open field on days 3 and 10. The open field is an unfamiliar environment to the rat that naturally tends to explore novel situations. A rat that is sedated or ill will travel less when exposed to the new environment such as the open field, which allows for rat locomotion a nd motor activity (Vogel, 2002). In contrast, an animal that is stimulated and healthy will spend more time explor ing the center of the open field and will travel further. During the latter part of the open fiel d test, technical difficulties prevented computer analysis of data from nine animals per trea tment. Greater treatment replication may have amplified the numerical differences reported. The fact that none of the Mucuna treatments affected behavior in the open field on day 3 or 10 may have been because of the relatively low Mucuna inclusion rate and also because oral ad libitum L-Dopa administration produces a more gradual and lower maximum concentration 87

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A: Alkaline phosphatase Figure 5-3. Effects of feeding detoxified Mucuna pruriens on blood levels of A) alkaline phosphatase; B) phosphorus; and C) m onocytes. CON = control diet; MUC = untreated Mucuna diet; ACD = Mucuna beans detoxified by acetic acid extraction; BAS = Mucuna beans detoxified by sodium hydroxide extraction; SIL = Mucuna beans detoxified by ensiling. Means wit hout a common superscr ipt letter differ ( P < 0.05); error bars denote standard errors. B: Phosphorus C: Monocytes ab a a a CON MUC ACD BAS SIL 0 50 100 150 200 250 300 350U/L b CON MUC ACD BAS SIL 0.0 2.5 5.0 7.5 10.0 12.5mg/dl a b a a a 0.40 0.35 0.30 0.25 0.20 0.15 0.45x103/ul b a ab ab b 0.10 0.05 0.00 SIL CON MUC ACD BAS 88

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(Cmax) in the plasma (Bartus et al., 2004). Rats were fed ad libitum and thus continuously exposed to L-Dopa. The numerically greater distance travelled and line crossings of rats fed ACD and SIL versus MUC on day 10 suggest that detoxification procedures that acidify the beans had less adverse effects on behavior. Both ACD and SIL had lower pH (pH 3.0 and pH 4.5 respectively) than BAS (11.0) and MUC (6.2). The lower pH may have played a role in reducing adverse effects of the MUC diet on behavior. The trend toward less abnormal behavior in rats fed ACD and SIL could also be partially due to their lower L-Dopa concentrations relative to MUC. Despite the relatively low dietary Mucuna inclusion rate of 10% DM and the ad libitum feeding, the total distance travel ed and total line crossings for days 3 and 10 were reduced by feeding Mucuna diets, indicating increased abnormal be havior relative to CON. Abnormal behavior in rats fed MUC may be related to side effects caused by Mucuna -derived hallucinogens (Szabo and Tebbett, 2 002). Cotzias et al. (1974) repor ted that feeding rodents high levels of L-Dopa adversely aff ected behavior and caused motor hyperactivity, muscle jerks, and stereotyped movements. Intake of L-Dopa in humans is also associated with psychiatric disturbances, which can lead to nervousne ss, anxiety and agitati on, insomnia, confusion, delirium, depression as well as psychotic reactio ns with hallucinations and anorexia (Szabo and Tebbett, 2002). However, because the reductions in total line crossing and distance travelled also occurred in detoxified Mucuna diets, they may have been due to components other than L-Dopa. According to Sato et al. (1994), chronic LDopa exposure is often associated with a gradual decline in efficacy. Murata (2006) re ported that Parkinsons disease patients who showed wearing-off phenomenon had higher Cmax and shorter L-Dopa half life (T1/2) than patients not displaying wearing-o ff. This suggests that over ti me neurotoxic signs such as confusion and agitation would give way to peripheral toxicity su ch as gastrointestinal upset 89

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(reduced intake or anorexia). Although feed intake was not reduced during this study, numerically lower travel and line crossings in rats fed ACD and SIL versus MUC on day 10 may have been signs of the initial stage of neur otoxicity (confusion, nervousness, depression). The fact feed intake and weight gain di d not differ among treatments, suggests that acceptability and nutrient bioavailability of control and Mucuna -based diets were similar. Mucuna-based diets have reportedly been associated with a decrease in acceptability and intake compared to soybean based diets (Del Carmen et al., 1999; Flores et al ., 2002). The relatively low Mucuna inclusion rate (10% of diet DM) in this study may explain why intake was not affected by feeding MUC in this study. Although the detoxification methods resulted in different L-Dopa and CP concentrations, similar performance and clinical data suggest the CP bioavailability and food safety were comparable among detoxified treatment diets. So lvent extraction typically disrupts the protein structure and degrades AA in Mucuna (Adebowale et al., 2007), ne vertheless, feeding BAS and ACD did not adversely affect growth and perfor mance, likely due to the relatively low dietary Mucuna inclusion rate. Adverse effects due to MUC consumption evidenced by splenomegaly and monocytosis were not evident when detoxified diets were fed. The splenomegaly caused by MUC agrees with studies where spleen enlargement occurred when poultry was fed Mucuna beans (Iyayi and Taiwo, 2003; Iyayi et al., 2005; Pugalenthi et al., 2005; Carew and Gernat, 2006). The spleen is the largest collection of lymphoid tissue in the body and splenomegaly resulting from feeding MUC probably reflects increased workload or hyper-function of the organ. Splenomegaly is associated with red blood cell destruction in the spleen, congestion due to portal hypertension and infiltration by leukemias and lymphomas, obstruction of blood flow or antigenic stimulation, 90

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and infection (Grover et al., 1993). Carew et al (2003) observed lymphoid necrosis, macrophage proliferation and lympho-phagocyt osis of the spleen at a 12% Mucuna inclusion in the diet of broilers. Iyayi et al. (2005) re ported that lymphoid depopulation in the spleen is indicative of the degenerative effects associated with feeding raw Mucuna beans. Relative to CON, the dietar y inclusion of undetoxified Mucuna bean (MUC) also caused monocytosis, a state of excess monocytes in the peripheral blood indicative of various disease states. Monocytes are leukocytes that replenish macrophages and dendritic cells and elicit an immune response at infection sites. In the tissu es, monocytes mature in to different types of macrophages that are responsible for phagocyt osis of foreign substances in the body. Monocytosis can indicate inflammation, stress du e to disease, hyperadrenocorticism, immunemediated disease, and malignant tumors (Meuten, 2008). The immediate causes of splenomegaly and m onocytosis in the cu rrent study are not clearly evident though both conditions reflect a response to a clinical condition. Interestingly, differences with respect to spleen weight a nd concentrations of phosphorus and monocytes counts between rats fed MUC versus CON were not evident when those fed CON versus detoxified diets were compared. Since the detoxified diets cont ained reduced levels of L-Dopa, the main toxic compound of concern in Mucuna, it is likely that L-Dopa toxicity was at least partially responsible for these clinical conditions in rats fed MUC. Alkaline phosphatases remove phosphate group s by dephosphorylation, and they are most effective in an alkaline environment (Coleman, 1992). Feeding undetoxified Mucuna resulted in lower plasma alkaline phosphatase (hypophospha tasemia) and phosphorus concentrations relative to detoxified Mucuna treatments. Phosphatases are involved in signal transduction because they regulate the action of proteins to wh ich they are attached (Steelman et al., 2008; Yi 91

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and Lindner, 2008). To reverse the regulatory effect, phosphate is removed on its own by hydrolysis or through mediation of protein phosphatases. Hypophosphatasemia can occur in cases of malnutrition, hypothyroidism, anemia, and chronic myelogenous leukemia. Given the fact that diets were manufact ured by a renowned feed company specialized in rodent diets, Harlan Teklad, and formulated to supply simila r quantities of nutrients it is not likely that malnutrition caused the hypophospatasemia. In support of this statement, rats were fed ad libitum and no differences in feed intake among treatments were observed. The rats were not anemic at the time of necropsy as shown by their red blood cell counts (7 x 106/ul) that were within the normal range (3-8 x 103/ul) as indicated by the cl inical pathology laboratory. Conclusion It can be concluded that dietary incl usion of detoxified or undetoxified Mucuna at 10% of diet DM did not affect any performance meas ure. Compared to feeding CON, feeding MUC decreased blood phosphorus concentration and ca used splenomegaly and monocytosis but feeding detoxified Mucunabased diets did not have these effects. Feeding MUC also decreased alkaline phosphatase levels re lative to feeding detoxified Mucuna diets. Therefore, the detoxification processes improved the safety of Mucuna. The behavior of rats fed all Mucuna diets instead of CON was characterized by decreased total travel distance and decreased total line cr ossings. These reductions were numerically less pronounced in rats fed ACD and SIL versus MUC or BAS in an open field test, suggesting that these detoxification methods are more promising than the BAS method. This aspect of the study was partly compromised by an equi pment problem. Therefore, future research should repeat the open field test to provide more conclu sive results on effects of detoxifying Mucuna on the behavior of rats. 92

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Because feeding detoxified Mucuna increased the concentration of alkaline phosphatase, which is a key enzyme regulating gene expressio n, future research should examine effects of feeding Mucuna on gene expression. Follow up research s hould also focus on long term effects of feeding the detoxified diets to multiple monogastric species while taking into account that a larger number of animals (n>12) may allo w better detection of treatment effects. 93

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CHAPTER 6 EFFECT OF FEEDING Mucuna pruriens OR LEVAMISOLE INJECTION ON Haemonchus contortus INFECTION IN LAMBS Introduction Infections caused by gastrointe stinal nematodes are the major constraint in small ruminant production. A helminth of particular concern, esp ecially in tropical and sub-tropical regions worldwide, is Haemonchus contortus The considerable egg-laying ca pacity of this nematode is maintained by adults feeding on blood. The late stage immature larvae also feed on blood. Blood loss can result in anemia, anorex ia, loss of condition, and eventu al death in the host animal (Miller and Horohov, 2006). Haemonchus contortus infects sheep, goats, deer, and other small ruminants and has been a significant cause of economic loss to small ruminant producers worldwide (Lange et al., 2006). The prophylaxis of haemonchosis has been jeopardized by selection for nematodes with resistance to anthelmintics (Bricarello et al., 2005). Extensive use of anthelmintics for the control of helminth infections has resulted in drug resistance, which is usually manifested by poor clinical response to anthel mintic treatments, though the latte r may also be due to other factors. Taylor et al. (2002) described several other conditions that may cause clinical signs similar to those normally associated with pa rasitism as well as a variety of reasons why anthelmintics fail to control nematodes. Due to the increasing problem of parasite resistance to antiparasitic drugs and the increased concern about drug residues in animal products and the environment, the application of nutraceutical and other biological approaches to parasite control has become more urgent. Ethnoveterinary sour ces have advocated exploiting antiparasitic properties of plant secondary metabolites with prophylactic or therapeutic properties (Athanasiadou and Kyriazakis, 2004). Further incentiv es to investigate alternative solutions are the economic loss that occurs due to decr eased production, the cost s of prophylaxis and 94

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treatment, and the deaths of valuable livestock. Recently several studies have been conducted to study alternative methods of internal parasite control (Albonico, 2003; Kerboeuf et al., 2003; Bricarello et al., 2005; Louvandini et al., 2006). Mucuna pruriens is a legume indigenous to tropical regions, especially Africa, India, and the West Indies. Szabo and Tebbett (2002) stated that the major drawback of Mucuna, which has compromised its usefulness as a food/feed s ource for humans and ot her monogastrics is associated with its L-Dopa concentration. Mucuna however, has been successfully used in the diet of ruminants that can digest the L-Dopa it contains (Mui nga et al., 2003, Mendoza-Castillo et al., 2003, Matenga et al., 2003; Chikagwa-Malunga et al., 2008b). Mucuna beans contain varying levels of L-Dopa (S t-Laurent et al., 2002; Daxenbich ler et al., 1972; Tebbett, 2002; Szabo and Tebbett, 2002). One study reported as little as 1.5% to as much as 9% L-Dopa in the bean of M. gigantea in southern India (Rajaram and Janardhanan, 1991). Sources from various countries claim that Mucuna has anthelmintic properties (Taylor, 2004) but there is only anecdotal evidence to support this claim. Research in which Mucuna was substituted for soybean meal in the ration of sheep (Chikagwa-Malunga et al. 2008d) indicated lower coccidial oocyst scores ( P < 0.05) and a 52% numerical re duction in fecal egg counts (FEC) in lambs fed a high Mucuna diet. When Jalalpure et al. ( 2007) applied ether-extracted oil from Mucuna pruriens beans to intestinal worms, they reported a significant increase in the paralysis of worms. Conroy and Thakur (2005) reported that Mucuna pruriens trichomes treatment was as effective against gastro-intestinal parasites in pregnant does as the commercial anthelmintic medicine fenbendazole. These resu lts emphasize the need for further scientific investigation of the anth elmintic properties of Mucuna. The aim of this study was to determine if incorporation of Mucuna beans in the diet reduces helminth parasite infection in lambs. 95

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Materials and Methods Animals The study was conducted during the summer of 2 007 at the University of Floridas sheep research unit, Gainesville, FL, USA (30o N lat.). The investigati on involved a 7-week trial. Thirty-six, 6-month-old, Dorper x Katahdin ram lambs weighing 28.8 + 5 kg were de-wormed subcutaneously with levamisole (0.4 mg/kg), balanced for body weight and FEC, and randomly allocated to three treatment groups. The 12 la mbs in each treatment group were randomly assigned to four pens, each with in one of four blocks representing different areas of the barn. Each pen contained three randomly chosen la mbs from a specific treatment. Pen was the experimental unit for feed intake, but sheep wa s the experimental unit for other measurements. The randomized complete block design consisted of 12 lambs in each treatment group, randomly assigned to four blocks, with three lambs in each pen. Treatments Mucuna pruriens cv. Aterrima beans were imported from Brazil (Wolf & Wolf Seeds Inc. FL, USA). The beans contained 31% CP and an L-Dopa concentration of 5.3%. All lambs were fed ad libitum amounts of total mixed rations formulat ed to be isonitrogenous (14% CP) and isocaloric (64% total digestible nutrients). The control diet contai ned 44% corn grain, 17% cottonseed meal, 0% Mucuna meal, 34% cottonseed hulls, and 3% molasses (dry matter (DM) basis). The Mucuna diet contained 53% corn grain, 0% cottonseed meal, 36% Mucuna meal, 6.5% cottonseed hulls, and 3% molasses (DM basi s). Treatments consisted of lambs fed 1.) the control diet (CON) and no anthel mintic, 2.) the diet in which Mucuna replaced cottonseed meal as the main protein source with no anthelmintic (MUC), and 3.) the control diet with weekly subcutaneous injections of levamisole (0.4 mg/kg; ANT). Lambs were allowed a 2-week 96

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adaptation period to adjust to their new diets before e xperimental challenge with H. contortus larvae for 3 weeks. Animals were kept on the assi gned regimen for a total of 5 weeks. Subsequently, two lambs per pen were harvested for dressing es timation and the third lamb was grazed on bahiagrass pasture for 19 days and then harv ested for abomasal worm counts and dressing estimation. Figure 6-1. Experimental conditions for anthelmintic trial: A) Cu stomized diaper designed to collect H. contortus eggs from a donor goat; B) the Baermann technique was used to harvest larvae before microscopically identifying and quantifying them. Haemonchus contortus Challenge Daily fecal outputs were collected from a na turally infected donor goat with fecal egg counts of up to 1900 eggs/g using a customized co llection diaper (Figure 6-1). Fecal matter was incubated at 37oC in pans covered with cheesecloth for a 2-week period. During incubation, regular moistening with double distilled water prevented drying, and kneading provided 97

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additional aeration. Microscopic identification and quantifica tion confirmed an abundance (>200/ml) of infectious L3 larvae. Larvae were ha rvested from the fecal matter (Figure 6-1) via the Baermann technique (Baermann, 1917). Larvae were stored under a thin layer of water in culture flasks at room temperatur e. During the third through the fi fth week of the trial, animals were challenged 3 times per week by gavage (Figure 6-2) with infectious H. contortus larvae (2000 larvae/lamb in 10 ml of distilled water). A B Figure 6-2. Introduction of in fectious larvae from donor goat through gavage: A) Haemonchus contortus eggs were collected from a donor goat, Jack, then incubated and larvae cultured; B) Lambs were dosed by gavage with resulting infective-stage larvae. Measurements Animal performance measures included feed intake, body weight gain, and dressing. Measurements were taken to monitor both anim al production and clinic al status. Clinical evaluation involved weekly FEC and anemia sc oring with abomasal worm counts (AWC) upon necropsy. For FECs, feces were collected directly from the rectum of each lamb and analyzed using the McMaster technique (Lan ge et al., 2006). To test for an emia (Figure 6-3), the color of the ocular conjunctivae was scored using the FAMACHA Anemia Guide (Kaplan et al., 2004). In addition, 7 ml of blood was collected by j ugular venipuncture into vaccutainer tubes 98

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containing EDTA for refractometric analysis of blood protein levels Capillary tube specimens of blood were spun in a microhematocrit centrifuge to determine packed cell volume (PCV). Figure 6-3. Anemia indicators were used to determine clinical signs of haemonchosis: A) Blood collection by jugular venipuncture and ex amination of the ocular membrane for FAMACHA scoring, B) Blood protein was m easured by refractometer and packed cell volume determined after spinning capil lary tubes in a microhematocrit centrifuge. Statistical Analysis The experimental design was a randomized comp lete block with 36 lambs, four blocks, three treatments, 12 pens, and three lambs per pen. The models used to analyze individual treatment effects were: For one time measurements: Yij = + Ti + Bj + Eijk Where: Yij = dependent variable = general mean Ti = treatment effect (nutritional measurem ents, abomasal worm counts, dressing) Bj = block effect (random effect) Eijk = experimental error 99

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For weekly measurements: Yijk = + Ti + Bj + TBij + Wk + TWik + BWjk + TBWijk +Eijkl Where: Yij = dependent variable = general mean Ti = treatment effect (FEC, PCV, blood prot ein, FAMACHA score, weight gain, fixed effects) Bj = block effect (random effect) TBij = treatment x block interaction Wk= week (repeated measurement) TWik = treatment x week interaction BWjk = block x week interaction TBWijk = treatment x block x week interaction Eijkl = experimental error Significance was declared at P < 0.05 and tendencies at P < 0.10. Statistical analysis was performed with the MIXED model proce dure of SAS (SAS 9.1, SAS Inst. Inc., Cary, NC, USA). The GLIMMIX procedure from SAS was used to analyze counts of fecal eggs. Significance was declared at P < 0.05. Results Clinical Measurements All lambs developed mature egg-laying worms (Figure 6-4). The ANT treatment decreased FEC and AWC by 87 and 83% respectively, relativ e to the CON treatment. The MUC treatment did not statistically affect FEC or AWC though a numerical reduction ( P > 0.1) was evident. As illustrated in Figure 6-5, neither ANT nor MUC treatment affected packed cell volume (32.4%), blood protein concentration (6 g/dL), or average FAMACHA score (2) and sheep weights were not affected by treatment (Figure 6-6). 100

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B A H contortus Abomasal Fecal egg counts Figure 6-4. Effect of feeding Mucuna (MUC) versus feeding a cont rol diet without (CON) or with subcutaneous levamisole anthelmin tic (ANT) treatment on A) fecal egg counts and B) H. contortus counts in the abomasums. Ba rs without a common superscript letter differ ( P < 0.05); error bars denote the standard error. Performance Measurements Daily feed intake, final body weight, average daily gain, and dressing were not different across treatments (Table 6-1). On averag e, body weight was higher for ANT (41.5 + 1.1 kg) than for MUC (37.6 + 1.1 kg) treatment groups, but MUC and CON (39.4 + 1.1 kg) had similar body weights and body weights did not differ between treatments during successive weeks (Figure 66). Table 6-1. Effect of feeding Mucuna versus Levamisole injection on performance of lambs Control Levamisole Mucuna SEM Initial weight, kg 28.7 28.7 28.8 1.5 Intake, %BW 5.0 5.2 5.6 0.4 Intake per pen, kg/d 5.8 6.3 6.5 0.4 Final weight, kg 43.1 44.9 41.5 1.5 Total weight gain, kg 14.5 16.2 12.7 1.2 Daily gain/lamb, kg 0.3 0.4 0.3 0.1 Dressing 1A, % 49.1 48.6 49.6 1.8 Dressing 2B, % 47.0 50.3 45.8 1.9 Means within each row with different superscripts are different, P < 0.05; SEM = standard error of mean. A Dressing of lambs fed the total mixed ration alone for 5 weeks. B Dressing of lambs grazed on pasture after the 5-week total mixed ration feeding regime. CON ANT MUC 0 100 200 300 400 500 600 FEC (eggs/g)b b a CON ANT MUC 0 500 1000 1500b b aworm counts/abomasu m 101

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25.0 30.0 35.0 40.0 123456Packed cell volume (% ) ANT CON MUC A 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 123456 Time (weeks)Blood protein (g/dl) B Figure 6-5. Effect of feeding Mucuna (MUC) versus feeding a cont rol diet without (CON) or with (ANT) subcutaneous levamisole anthelmintic treatment on A) packed cell volume (pooled SEM = 0.5%) and B) blood protein (pooled SEM = 0.1 g/dl) measurements at various time poi nts; NS = not significant ( P > 0.05). Treatment and treatment x time effects were not significant, P > 0.05; time effect was significant, P < 0.05. 102

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25.0 30.0 35.0 40.0 45.0 50.0 1234567 Time (weeks)Sheep weight (kg) ANT CON MUC Figure 6-6. Effect of feeding Mucuna (MUC) versus feeding a cont rol diet without (CON) or with (ANT) subcutaneous levamisole anthelmintic (ANT) treatment on sheep weights (pooled SEM = 1.1 kg) at various time points. NS = not significant (P > 0.05); treatment and treatment x time effects were not significant, P > 0.05; time effect was significant, P < 0.05. Discussion Neither levamisole treatment nor feeding Mucuna affected anemia indicators. Even with the experimentally induced abomasal worm bur den, which resulted in up to 1400 adult worms per lamb, no signs of anemia or adverse affects on animal productivity performance were observed. Roberts and Swan (1982) investigated the correlation between worm burden and anemia. Their findings suggest that the number of worms associated with low hemoglobin (Hb) levels varied with the bodyweight of th e sheep. For sheep up to 20 kg, 10.5 g% Hb was associated with 112 worms and 8 g% with 355 worms. However, 355 worms caused only moderate depression of Hb levels in sheep over 50 kg, and 1259 worms were required to cause severe depression (< 8.0 g%) in sheep over 50 kg In the current study av erage animal weights were less than 50 kg suggesting that the worm burden of up to 1400 worms would have been sufficient to cause severe Hb depression and anemia. Some sheep in poor condition and kept 103

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under poor grazing conditions can be severely anemic in the presence of less than 100 worms (Roberts and Swan, 1982). This suggests that the clinical signs of infection were likely obscured by a combination of factors such as a high plane of nutrition and utilization of lambs of breeding known to be somewhat resistant/resilient to this parasite. Th e Katahdin is a hair-type sheep developed in the United States from West African hair sheep a nd wooled British sheep while the Dorper was developed in South Africa from the Dorset Horn and Blackheaded Persian for use in arid areas. Dorper sheep have low resistance to parasites, bu t are able to cope with infection by maintaining their PCV levels and body condition; in contrast Katahdins are more resistant, showing lower FEC than Dorpers (Vanimisetti et al., 2004). Breed differences are more apparent when infection levels are higher and animals are less affected when on a better plane of nutrition. Dietary CP and metabolizable protein (MP) espe cially impact susceptibility to H. contortus Sheep fed moderate (10.2% CP, 75 g MP) or high (17.2% CP, 129 g MP) levels of dietary protein responded differently to helminth infection (Bricarello et al., 2005) The higher MP supply resulted in higher body weight gains and PCVs ( P < 0.05) in addition to an ability to withstand the pathophysiologi cal effects of H. contortus Optimal nutrition thus increases resilience to H. contortus -induced pathophysiology (Bricarello et al., 2005). In the current study the dietary CP concentration was about 14% of DM and this high CP supply may have conferred some resistance to haemonchosis. Kahn et al. (2003a, b) noted that increased immunity and resistance as a result of protein supplementation is de pendent on the prevailing supply and demand for scarce nutrients such as protein. Knox and Steel (1999) suggest that ev en urea supplementation can increase resilience to parasitism, thereby improving performance and enhancing resistance mechanisms against worms in sheep on low quality diets. According to Datta et al. (1999), 104

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animals fed high protein diets showed highe r performance, higher antibody responses to H. contortus antigens, and reduced fecal e gg counts. In future research, Mucunas anthelmintic potential will need further evaluation under less optimal nutritional conditions. In the current study, the lack of pathologi c responses in spite of the presence of considerable numbers of adult abomasal worms and fecal egg output as well as evidence of abomasal hemorrhage (Figure 6-7), indicates that the usefulness of anemia detection methods, such as PCV, blood protein, and FAMACHA scori ng to monitor internal parasite burdens is limited to situations with low levels of nutrition and poorer body conditions in less resistant/resilient breeds. Mucuna treatment had no clinically significant an thelmintic effect compared to the controls. Levamisole is a broa d-spectrum anthelmintic drug wi dely used to reduce parasitic nematode burdens in livestock, but the form erly high efficacy of levamisole against H. contortus in sheep and goats has been compromised by the development of resistance in field populations; this resistance occurs at a slower pace than with other classes of anthelmintic drugs (Conder et al., 1991). However, the fact that levamisole treatment did not reduce FEC or AWC by more than 87% and 83% respectively, suggest s that the worms in this study were partially resistant to levamisole. Haemonchus contortus strains that are considered resistant provide levamisole efficacy rates as high as 77% as opposed to 100% efficacy with drugs that are considered effective (Uppal et al., 1993). Some literature suggests that the anec dotal anthelmintic properties of Mucuna are in the hairs on the pods (Conroy and Thakur, 2005). In some varieties, these hairs or trichomes cause a burning/itching sensation upon touch, wh ich can be attributable to serotonin, mucunain or some other compound (Szabo and Tebbett, 2002). Szab o and Tebbett (2002) screened for toxic 105

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compounds, but only reported serotonin to be pres ent in the trichomes. Ot her literature suggests the L-Dopa concentration to be responsible for possible anthelmin tic effects (Faridah Hanum and van der Maesen, 1996). Chikagwa-M alunga et al. (2008a) report the L-Dopa concentration of the Mucuna plant to be mostly concentrated in the b eans and the pods and these authors showed that replacing soybean meal with Mucuna reduced coccidia scores significantly and numerically reduced FEC. Therefore more studies on the anthelmintic effects of Mucuna trichomes versus beans are needed. Although the main focus of this experiment was to investigate the anecdotal claims about anthelmintic properties of Mucuna, another very important consid eration is the potential for adverse effects of Mucuna ingestion on animal health and pr oduction. According to Githiori et al. (2006), Ketzis et al. (2006) and Athanasia dou et al. (2001), dose-depe ndent anti-parasitic properties and cost effectiveness need to be carefully determin ed, because some of the active compounds of plants with anti-parasitic proper ties may also have antinutritional effects which may lead to reduced feed intake and poor perf ormance. Several adverse effects have been associated with Mucuna intake in monogastrics (Del Carmen et al., 1999; Carew et al., 2002; Del Carmen et al., 2002; Flores et al., 2002), most of which are attributable to L-Dopa intake including reduced feed intake, weight loss, and feed conversion efficiency, diarrhea, vomiting, and skin lesions (Del Carmen et al., 2002; Flor es et al., 2002). In c ontrast, ruminants are seemingly not adversely affected by intake of Mucuna L-Dopa. This is partly because up to 53% of dietary L-Dopa can be digested in rumi nal fluid (Chikagwa-Malunga et al., 2008c). Other studies showed that feeding Mucuna improved DM intake, weight gain and milk production in ruminants without detrimental effects (Burgos et al., 2002; Muinga et al ., 2003; Eilitta et al., 2003; Matenga et al., 2003; Mendoza-Castillo et al., 2003; Nyambati and Sollenberger, 2003; 106

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Perez-Hernandez, 2003). In th e present study the mean daily feed intake (2.5 kg), final body weight (37.9 kg), average daily gain (0.31 kg/d) dressing (48.8%) were not different across treatments. This indicates that inclusion of 36% Mucuna in the diets did not adversely affect performance of the lambs, and suggests th at in future research, the level of Mucuna in the diet could be increased to further re veal anti-parasitic properties. Figure 6-7. Upon necropsy H. contortus worms were harvested from the abomasums and quantified: A) Abomasal content was sc reened for worms and representatively subsampled; B) Necropsy revealed abomasal hemorrhage and internal bleeding; C) Subsamples from the abomasal content we re collected in petri dishes and worms counted on a light box. A B C Conclusion It can be concluded that Mucuna bean intake did not reduce H. contortus infection in lambs fed at a high plane of nutri tion. In the current study, feeding Mucuna did not affect fecal egg counts or abomasal worm counts, though a numerical ( P > 0.10) reduction was evident at the 36% Mucuna inclusion rate. Future studies should inve stigate whether higher levels of dietary 107

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Mucuna inclusion result in anthelmintic respons es and determine if the 36% dietary Mucuna inclusion level reduces helminth infec tion in lambs fed poorer quality diets. 108

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CHAPTER 7 GENERAL SUMMARY, CONCLUSI ONS AND RECOMMENDATIONS Mucuna pruriens is a tropical legume, a versatile cover crop attractive as a green manure for sustainable farming systems, a promising prot ein and starch supplement for the food and feed industry, as well as a potential source of nutraceutical compounds. However, Mucuna also contains toxic secondary compounds, particularly L-Dopa, which reduce its appeal as a dietary ingredient for humans and monogastric livesto ck. Interestingly, th ese toxic compounds reportedly contribute to Mucunas effectiveness as a nutraceutical such as in the treatment of Parkinsons disease. Several addi tional anecdotal claims about Mucunas healing properties exist, but many of these require further scientific validation. In ruminant feeding practice Mucunas toxicity has not posed a problem. Rather, Mucuna supplementation to ruminants increased nitrogen intake and retention, we ight gain, and milk production in ruminants (Eilitta et al., 2003). Un til recently, the reason why ruminants are less susceptible to the negative impact of Mucunas high L-Dopa concentration was not known. Recent evidence indicates this is because the L-Dopa is degraded in the rumen (ChikagwaMalunga et al., 2008b), theref ore, it is subsequently not presen t in the muscle tissue or blood of animals fed Mucuna-based diets at high concentrations. Conversely, studies have shown that humans who consume high levels of undetoxified Mucuna may experience anorexia, confusion, nausea, diarrhea, and vomiting. Research ha s also shown that feeding undetoxified Mucuna to monogastric livestock reduces intake, body weight and feed conversion effi ciency, and increases mortality. Some studies indicate that various processing techniques can reduce the L-Dopa concentration in Mucuna beans to safe levels (< 0.4%), but these techniques are often costly, time consuming, or require scarce resources. Few studies have examined the effects of feeding 109

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detoxified Mucuna to monogastrics and little is known about their impact on the nutritional value of the bean. Four studies were conducted to address some of these unknowns. The first study examined the effect of ensili ng duration on the fermentation of Mucuna and how ensiling affects the L-Dopa content and nutritiona l value of the bean. Subseque nt detoxification experiments removed L-Dopa through sonication, or acid or al kaline solvent extraction, and investigated how these methods affect its nutritive value. Anot her experiment examined the performance, physiological and behavioral changes of Sprague -Dawley rats fed undetoxi fied or detoxified beans. The last experiment investigated how ingestion of Mucuna beans affected helminth parasite infection in lambs. Further details on e ach of these experiments are summarized below. The objective of Experiment 1a) was to exam ine how long it takes to decrease the pH of ensiled Mucuna to 4.5. Crushed beans (6 mm) were ensile d in the dark at room-temperature (18 to 25oC) for 0, 3, 7, 21, and 28 days. A pH of 4.5 a nd an L-Dopa concentration of 1.3% (54% reduction) were recorded after 28 days of ensilin g. The objective of Experiment 1b) was to study the effect of partic le size of ensiled Mucuna on L-Dopa concentration and on fermentation and nutritional characteristics. Mucuna beans were ground to pass through 2, 4, and 6 mm screens and ensiled for 28 days. Ensiling decreased the LDopa concentration of 2, 4 and 6 mm particles from 2.8% to 1.2, 1.6, and 1.1%, respectively. Ensili ng also reduced the WSC concentration and pH and increased NH3N concentration. Neither en siling nor particle size a ffected concentrations of fat (5%), crude protein (23-25% ), starch (38-40%), or neutra l detergent fiber (17-20%). Dry matter losses (< 1%) and mold or yeast counts were unaffected by pa rticle size. Aerobic stability was maintained beyond 657 hours in all treatments. The total-acid concentration of 2, 4, and 6 mm particles contained 54, 58, and 46% lactate, respectively. Theref ore the lactate:acetate ratio of all samples exceeded 3.0. In conclusion, ensiling Mucuna bean for 28 days reduced the L110

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Dopa concentration by 43 to 61% while preserving most nutrients. Coarse and fine particles had similar effects on nutritional composition, fermenta tion indices and extent of L-Dopa removal, indicating that grinding is unnece ssary for ensiling to be effective. Further research should examine the L-Dopa concentra tion and nutritive value of crac ked (by blunt force) versus coarsely crushed beans, as cracking require s less energy. The ensiling experiments were terminated after 28 days due to achievement of the typical minimum pH for legume silages. Future research should examine whether ensiling Mucuna beans for periods in excess of 28 days will further reduce the pH and the L-Dopa c oncentration. During the current process, an automated vacuum-sealer was used to ensure an anaerobic environment in the bags. Future research should investigate if similar results are obtained when Mucuna is ensiled in conventional plastic bags without the vacuum air exclusion step. In Experiment 2, the goal was to study the e ffects of three extraction methods on L-Dopa concentration and nutritional composition of finely(1 mm) or coarsely(6 mm) ground Mucuna beans. Methods included extracti on in solutions of acetic acid (A CD, pH 3) or sodium hydroxide (ALK, pH 11) for 8 hours or sonication (SON) fo r 5 minutes. All three methods reduced L-Dopa concentrations of fine Mucuna particles from 2.8 to < 0.2% and increased NDF and starch concentrations by at least 62 and 14%, respectively. Fat concentration of fine particles was reduced from 5.5% to 4.2% by SON but not by AC D and ALK. The methods also reduced CP and WSC concentrations of fine particles by 24-31% and 78-81%, respectively. Sonication and ACD did not reduce L-Dopa concentration of co arsely ground beans but ALK reduced it from 2.8 to 2.0%. Sonication reduced CP, WSC, and fat c oncentration of coarse particles by 6, 17, and 27%, respectively and ALK increased their star ch concentration by 17%. The ACD treatment increased the NDF concentration of coarse particles by 35% but ALK and SON did not; ACD 111

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and ALK reduced fat concentration by 31 and 35%. It was concluded that the extraction methods reduced the L-Dopa concentration of fine Mucuna particles to safe levels but increased their NDF and starch concentrations at the expense of their WSC and CP concentrations. Extraction methods were less effective at reducing the LDopa in coarse particles and had fewer, less consistent effects on their nutritional composition. The melanin concentrations in the detoxified bean particles need to be measured in future research because of concerns that they could predispose to melanoma. The maximum solubility of L-Dopa in acidic and alkaline solutions at pH of 3 and 11 needs to be determined in order to reduce the amount of solvent necessary to detoxify the bean particles. In addition, sonication in solutions other than water should also be investigated. Experiment 3 examined the effect of feeding detoxified Mucuna bean on the performance, behavior, and health of rats. Sixty Sprague-Dawley rats were randomly assigned to five treatments (n=12). Dietary treatments consisted of a commerc ial rat chow (CON) or diets in which 10% of a customized rat chow was repl aced with either untreated (undetoxified) Mucuna (MUC), or Mucuna detoxified by acetic aci d extraction (pH 3), sodium hydroxide extraction (pH 11), or ensiling for 28 days (SIL). During the cour se of the 14-day trial, behavior, physiological development, and signs of clinical pathol ogy were evaluated. Animals were necropsied afterwards. Feeding MUC caused splenomegaly and monocytosis, and reduced blood phosphorus concentrations relative to CON, but these effects we re not observed in rats fed detoxified diets. Feeding detoxified diets increased alkaline phosp hatase concentration by 11-17% compared to MUC, but not CON. No differences in performa nce or physiology were observed in any of the rats on the detoxified diets. Compared to CON, Mucuna-based diets gave similar feed intake and weight gain. When behavior was examined in the open field on days 3 and 10 individually, no 112

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abnormalities were observed; however, when the tota l distance traveled and total line crossings over the two days were taken into account, rats on all Mucuna -based diets showed decreased activity, yet this response tended to be less severe in rats fe d ACD and SIL treatments but not in MUC and BAS. It can be concluded that at the 10% level of dietary inclusion, there were fewer measurable adverse effects due to feeding the detoxified Mucuna bean compared to untreated Mucuna bean. The current research suggests that th e preferred detoxification method was ensiling for 28 days at 2 mm pa rticle size and 70% moisture. This method of ensiling was reasonably successful at reducing L-Dopa levels (57%), it allowe d a shelf life of at least 657 hours and preserved nutrients such as crude protein, starch, fat, and fiber (Chapter 3). This suggests superior nutritive value relative to acid or alkali solv ent extraction methods, which were more effective at reducing L-Dopa concentratio n but also decreased concentrations of key nutrients. Due to the equipment th at malfunctioned in the current study, the open field test should be repeated to better assess effects of the treat ments on behavior. Future studies should repeat this study on other monogastric species and compare effects of greater dietary inclusion levels of detoxified and undetoxified Mucuna. The aim of Experiment 4 was to determine if ingestion of Mucuna beans reduces helminth parasite infestation in lambs. Thirty-s ix Dorper x Katahdin ram lambs (six months old, 28.8 + 5 kg body weight) were dewormed subcutaneously with levamisole (2 ml/45.4 kg), balanced for fecal egg counts and body weight and randomly allocated to three treatment groups. The 12 lambs in each treatment group were randomly assigned to four pens, each containing three lambs. All lambs were fed ad libitum amounts of an isonitrogenous (14% CP), isocaloric (64% total digestible nutrients) total mixed ration in which the main protein supplement was cottonseed meal or Mucuna. Treatments consisted of a control diet, a diet in 113

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which Mucuna replaced cottonseed meal and a furthe r treatment that involved administering levamisole (2 ml/45.4 kg) to lambs fed the contro l diet. Lambs were adapted to diets for 2 weeks and trickle infected 3 times per week by gavage with infectious Haem onchus contortus larvae (2000 larvae/lamb) for 3 weeks. S ubsequently, two lambs per pen were necropsied and the third lamb was grazed on bahiagrass pasture for 14 d and then necropsied. All animals developed mature worms. Levamisole treatment decreased fecal egg counts by 87% (445 vs. 58 eggs/g) and abomasal worm counts by 83% (1170 vs. 202 worms/lamb). Mucuna intake did not affect fecal egg counts (445 vs. 412 eggs/g) or abomasal worm counts (1170 vs. 958 total worms), though a numerical (P > 0.10) reduction was ev ident. Neither levamisole nor Mucuna treatment affected anemia indicators [FAMACHA score (2), packed cell volume (32.4%) and blood protein concentration (6 g/dL)], daily feed intake (2.5 kg), final body weight (37.9 kg), average daily gain (0.31 kg/d) and dressing (48.8%). Mucuna intake did not reduce infection in lambs fed the high quality diet. Patholog ical signs of infection were obscure d, most likely by a combination of the high nutritional plane and lambs of breeding known to be at least somewhat inherently resistant to this parasite as compared to highl y improved breeds. Future studies should examine if Mucuna exhibits anthelmintic properties in more susceptible lambs fed poorer quality diets. This series of experiments show that Mucuna pruriens can be detoxified by means of solvent extraction in both acidic (pH 3) and alka line (pH 11) solutions. Both extraction methods are equally successful provided the bean particles are finely ground (1 mm) as this increases the surface interaction with the solv ent. The acidic extraction method is somewhat favorable over the alkaline method due to lesser discoloration of the bean. Sonication at the 1mm particle size level is, however, somewhat preferable to the solvent extraction methods because it resulted in similar L-Dopa removal from fine particles without di scoloration. Although successful in detoxifying 114

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the bean, these methods also reduced the CP a nd WSC concentration of the bean. In contrast, ensiling Mucuna beans for 28 days reduced the L-Dopa c oncentration to a lower extent, but did not affect the CP concentration of the bean, which is the primary reason for using Mucuna as a feed or food source. The ensiled bean had a long shelf life and ensiling did not cause discoloration of the bean. Furthermore, cour se and fine particles had similar L-Dopa concentrations and nutritive value. Ensiling is also less labor intens ive and more practical because grinding, washing, sieving and drying are not required. Therefore, ensiling is a more promising Mucuna detoxification method for resource-lim ited smallholders. Nevertheless, detoxifying Mucuna beans by acid or alkali solvent extrac tion or ensiling is recommended for monogastric diets because all of these methods prevented negative effects of feeding the undetoxified bean such as monocytosis and splenomegaly when the beans accounted for 10% of the diet of rats. The anthelmintic properties of Mucuna require further investigation under less optimal feeding conditions sin ce the high nutritional plane us ed in the anthelmintic study typically increases resilience to helmin ths and obscures pathological signs of H. contortus infection. 115

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LIST OF REFERENCES Adebowale, K.O. and Lawal, O.S., 2003a. Foami ng, gelation and electrop horetic characteristics of Mucuna bean ( Mucuna pruriens ) protein concentrates. Food Chemistry 83, 237. Adebowale, K.O. and Lawal, O.S., 2003b. Microstructure, physicochemical properties and retrogradation behavior of Mucuna bean ( Mucuna pruriens ) starch on heat moisture treatments. Food Hydrocolloids 17, 265. Adebowale, Y.A., Adeyemi, I.A., and Oshodi A.A., 2005a. Functional and physicochemical properties of flours of six Mucuna species. African Jour nal of Biotechnology 4, 14611468. Adebowale, Y.A., Adeyemi, A., and Oshodi, A. A., 2005b. Variability in the physicochemical, nutritional and antinutritional attributes of six Mucuna species. Food Chemistry 89, 37. Adebowale, Y.A., Adeyemi, I.A., Oshodi, A.A ., and Niranjan, K., 2007. Isolation, fractionation and characterisation of proteins from Mucuna bean. Food Chemistry 104, 287. Albonico, M., 2003. Methods to sustain drug efficacy in helminth control programmes. Acta Tropica 86, 233-242 Anonymous, 2002. http://www.wikipatents.com/6340474.html Association of Official Anal ytical Chemists (AOAC), 1984. Official methods of Analysis, Fourteenth edition, Washington DC, Procedure 24.005. Athanasiadou, S. and Kyriazakis I., 2004. Plant secondary metabolite s: antiparasitic effects and their role in ruminant production systems. Proceedings of the Nutrition Society 63, 631639. Athanasiadou, S., Kyriazakis, I ., Jackson, F., and Coop, R.L., 2001. Di rect anthelmintic effect of condensed tannins towards different ga strointestinal nematodes of sheep: in vitro and in vivo studies. Veterinary Parasitology 99, 205-219. Bachmann, T., 2005. Grassland and pasture crops: Grassland index Mucuna pruriens (L.) D.C. FAO, Rome. http://www.fao.org/ag/ AGP/AGPC/doc/GBASE/DATA/PF000416.HTM. Accessed: May 2008. Baermann, G., 1917. Eine einfache methode zur auffindung von ancylostoma-(nematoden-) larven aus erdproben, mededeel uithet gen eeskunde lab te Weltevreden, Feestbundel, Batavia. P 41-47. Balaban, M.O. and Teixeira, A.A., 2002. Potentia l home and industrial process treatments to reduce L-Dopa in Mucuna bean. In Food and Feed from Mucuna: Current Issues and the Way Forward. International Cover Cr ops Clearinghouse, Honduras, p. 339-351. 116

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Bartus, R.T., Emerich, D. Snodgrass-belt, P. F u, K., Salzberg-brenhouse, H., Lafreniere, D., Novak, L., Lo, E., Cooper, T., and Basile, A. S., 2004. A pulmonary formulation of L-Dopa enhances its effectiveness in a rat model of Parkinsons disease. Journal of Pharmacology and Experimental Therapeutics 310, 828-835. Bressani, R., 2002. Factors influencing nutritive value in food grain legumes: Mucuna compared to other grain legumes. In: B. M. Flores, M. Eilitta, R. Myhrman, L.B. Carew, R. J. Carsky (Eds), Food and Feed from Mucuna: Current Uses and the Way Forward. Proceedings of the Centro Internacional de Informacion sobre Cultivos de Cobertura (CIDICCO), Tegucigalpa, Honduras, p. 164-188. Bricarello, P.A., Amarante, A.F.T., Rocha, R.A ., Cabral Filho, S.L., Huntley, J.F., Houdijk, J.G.M., Abdalla, A.L., and Gennari, S.M., 2005. Influence of dietary protein supply on resistance to experimental infections with Haemonchus contortus in Ile de France and Santa Ines lambs. Veterinary Parasitology 134, 99-109 Bunch, C.C., 2002. Community-level development of Mucuna recipes: the example of Nutricocina. In Food and Feed from Mucuna: Current Issues and the Way Forward. International Cover Crops Clearinghouse, Honduras, p. 218-226. Burgos, A., Matmoros, I., Toro, E., 2002. Evaluation of velvetbean (Mucuna pruriens ) meal and Enterobium ciclocarpum fruit meal as replacements for soybean meal in diets for dualpurpose cows. In: B. M. Flores, M. Eilitta, R. Myhrman, L.B. Carew, R. J. Carsky (Eds), Food and Feed from Mucuna: Current Uses and the Way Forward. Proceedings of the Centro Internacional de Informacion sobre Cultivos de Cobertura (CIDICCO), Tegucigalpa, Honduras, pp. 228-237. Cameron, D.G., 1988. Tropical and subtropical pa sture legumes. Queensland Agricultural Journal March-April, 110-113. Capo-chichi, L.J.A., Eilitta, M., Carsky, R.J., G ilbert, R.A., and Maasdorp, B., 2003. Effect of genotype and environment on L-Dopa concentration in Mucunas seeds. Tropical and Subtropical Agroecosystems 1, 319-328. Carew, L.B., Hardy, D., Weis, J., Alster, F., Mi schler, S.A., Gernat, A., Zakrzewska, E.I., 2003. Heating raw velvetbeans ( Mucuna pruriens ) reverses some antinut ritional effects on organ growth, blood chemistry, and organ hist ology in growing chickens. Tropical and Subtropical Agroecosystems 1, 267-276. Carew, L.B., Valverde, M.T., Zakrzewska, E.I ., Alster, F.A., Gernat, D., 2002. Raw velvetbeans ( Mucuna pruriens ) and L-Dopa have differing e ffects on organ growth and blood chemistry when fed to chickens. In: B. M. Flor es, M. Eilitta, R. Myhrman, L.B. Carew, R. J. Carsky (Eds), Food and Feed from Mucuna: Current Uses and the Way Forward. Proceedings of the Centro Internacional de Informacion sobre Cultivos de Cobertura (CIDICCO), Tegucigalpa, Honduras, p. 272-287. Carew, L.B. and Gernat, A.G ., 2006. Use of velvetbeans, Mucuna spp. as a feed ingredient for poultry: a review. Worlds P oultry Science Journal 62,131-143. 117

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BIOGRAPHICAL SKETCH Christiaan Max Huisden (Max) was bor n on August 9, 1966 in the Dutch Caribbean (Curacao); he is a citizen of Suriname, South Am erica. His late parents, Max Franklin Huisden and Amoi Trude Leonie Huisden-Lie A Kwie, both citizens of Suriname, lived in Curacao for 24 years and all of their five children were born there. In 1989, Max attended the Agricultural Produc tion program of the Faculty of Technology at the Anton de Kom (ADEK) Universiteit van Suriname, and graduated in 1994 with his Bachelor of Science degree. After graduation he was employed as Department Head at the Palmoil and Animal Production company (GPOV) and as a Researcher by the Cent er for Agricultural Research (CELOS) in Suriname. In 1996 Max was awarded a fellowship by the Or ganization of American States (OAS) for a graduate program at the Univer sity of Floridas Animal Scien ces Department. Max received his Master of Science and Master of Agriculture degrees in Animal Science working respectively with Dr. Roger West in Meat Science and with Dr. Frank Simmen in Reproductive Physiology/Molecular and Cell Biology. From 2001 through 2004 he was employed by the Center for Environmental and Human Toxicology at the University of Florida as a Biological Scientist in Molecular Toxicology with Dr. Ev an Gallagher. During this time Max earned his certification as a Toxicologist and pursued a dual Doct orate in Holistic Nu trition (Ph.D.) and Naturopathic Medicine (ND) from Clayton College (Birmingham); he graduated with High Honors in 2006. In 2004 Max joined the Nutriti on laboratories of Dr. Adegbola Adesogan at the University of Floridas Animal Sciences Department as a Biological Scientist. Cu rrently Max is working

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toward certification in Drug Chemistry at the College of Pharmacy and the completion of a second Ph.D. degree, specializing in Nutrition and Pharmaceutics. As the point person for collaboration between the University of Florida and the ADEK Universiteit van Suriname, Max is currently sp earheading an effort to catalog and verify the medicinal properties of a myriad of functional foods and naturallyoccurring plant species in the Amazon rainforest. Max married his precious wife, Andrea Feaste r Huisden, in 2001 and they are blessed with four wonderful sons: Raoul Max Franklin Huis den (12), Christiaan Henry Huisden (5), John Franklin Sjaak Huisden (2), and Carlo Dennis Huisden (1).