THE EFFECTS OF FUNGAL STRESS ON THE SELECTED PLANT SEEDS AND ITS APPLICATIONS FOR NOVEL FOOD DEVELOPMENT FENG SHENGBAO (B Eng.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS I would like to express my deepest gratitude and heartfelt thank to my supervisor, Dr Huang Dejian, who gave great influence to me for his encouragement and invaluable advice all the way along His endless support and constructive criticism has been precious during these years I wish to thank my Co-supervisor, A/P Lee Yuan Kun, from Department of Microbiology, for his guidance during my Ph.D study My special thanks go to my former supervisor, A/P Philip J Barlow for his constant encouragement I would also like to thank the laboratory staff, Ms Lee Chooi Lan, Miss Lew Huey Lee and Miss Jiang Xiaohui who have helped extensively in the project Special thanks also go to Unicurd Food Company Pte Ltd (Singapore) for helping me with the constant supply of experimental materials during the first step of my postgraduate study The financial support from National University of Singapore is greatly appreciated II TABLE OF CONTENTS ACKNOWLEDGEMENTS II SUMMARY IX LIST OF TABLES XI LIST OF FIGURES XII LIST OF ABBREVIATIONS XVII LIST OF PUBLICATIONS XVIII LIST OF ABSTRACTS AND PRESENTATIONS CHAPTER INTRODUCTION XX 1.1 Background 1.2 Objectives CHAPTER LITERATURE REVIEW 2.1 Secondary Metabolites in Plants 2.2 Phytoalexins in Plants 10 2.2.1 Phytoalexins Definition 10 2.2.2 Mechanisms of Phytoalexins Production 12 2.2.3 Elicitors of Phytoalexins Generation 17 2.2.4 Phytoalexins and Plant’s Disease Resistance 19 2.3 Soybean Phytoalexin: Glyceollins 21 2.3.1 Glyceollins Biosynthesis 21 2.3.2 Biological Properties of Glyceollins 26 III CHAPTER FUNGUS-STRESSED GERMINATION OF BLACK SOYBEANS LEADS GENERATION TO OF OXOOCTADECADIENOIC ADDITION TO GLYCEOLLINS 3.1 Introduction ACIDS IN 29 30 3.1.1 Soybean and its Nutritioninal Values 30 3.1.2 Secondary Metabolites in Soybean 30 3.1.3 Black Soybean 32 3.1.4 Objectives 33 3.2 Materials and Methods 34 3.2.1 Materials and Instruments 34 3.2.2 Black Soybean Germination and Fungal Inoculations 35 3.2.3 Compound Identification and Isolation 36 3.3 Results 39 3.3.1 Fungal Growth in Germinating Black Soybeans 39 3.3.2 Fungal Stress to Germinate KODES and Glyceollins 40 3.3.3 Characterization of KODES and KODE Glyceryl Esters 47 3.4 Discussion 50 3.5 Conclusion 55 CHAPTER THE EFFECTS OF FUNGAL STRESS ON THE ANTIOXIDANT CAPACITY OF GERMINATING BLACK SOYBEANS 4.1 Introduction 4.1.1 Antioxidants in Soybean 56 57 57 IV 4.1.2 Oxidative Stress and Reactive Oxygen Species (ROS) Generation 59 4.1.3 ROS Catalyzed Lipid Peroxidation in Soybeans 60 4.1.4 Objectives 62 4.2 Materials and Methods 62 4.2.1 Black Soybeans Germination and Fungal Inoculations 64 4.2.2 Sample Preparation and Extraction 65 4.2.3 Analytical Methods 65 4.2.3.1 Vitamin E Analysis 65 4.2.3.2 Quantification of Lipid Hydroperoxides 66 4.2.3.3 Measurement of Antioxidant Capacity in the Hydrophilic Extract of Soybeans 67 4.2.3.4 Measurement of Antioxidant Capacity in the Lipophilic Extract of Soybean 4.2.3.5 Determination of Total Phenolics in Black Soybeans 70 73 4.2.3.6 Isoflavones Analysis in Hydrophilic Extract of Black Soybeans 74 4.3 Results 74 4.3.1 Vitamin E 74 4.3.2 Lipid Peroxides 75 4.3.3 ORACoil Value in Lipophilic Extract 75 4.3.4 Antioxidant 76 4.3.5 Total Phenolics Contents in Hydrophilic Extract 76 V 4.3.6 Isoflavones in Soybeans 79 4.4 Discussion 81 4.5 Conclusion 86 CHAPTER NOVEL PROCESS OF FERMENTING FUNGUS-STRESSED BLACK SOYBEAN [GLYCINE MAX (L.) MERRILL] YOGURT WITH DRAMATICALLY REDUCED FLATULENCE - CAUSING OLIGOSACCHARIDES BUT ENRICHED SOY PHYTOALEXINS 88 5.1 Introduction 89 5.1.1 Introduction of Yogurt 89 5.1.2 Nutritional Values and Health Benefits of Yogurt 90 5.1.3 Soy Yogurt and the Nutritional Values 93 5.1.4 Negative Effects in Soy Yogurt 95 5.1.5 Objectives 98 5.2 Materials and Methods 99 5.2.1 Materials 99 5.2.2 Black Soybean Germination under R oligosporus Stress 99 5.2.3 Soy Yogurt Fermentation 100 5.2.4 Isoflavones, Glyceollins and KODES Analysis 101 5.2.5 Sucrose and Oligosaccharides Analysis 102 5.2.6 Viable Bacterial Count 104 5.2.7 Titratable Acidity 104 5.2.8 Statistics 104 VI 5.3 Results 104 5.3.1 Soy Yogurt Fermentation 104 5.3.2 Sucrose and Oligosaccharide Contents 106 5.3.3 Isoflavones, Total Glyceollins and Total KODES 108 5.4 Discussion 113 5.5 Conclusion 115 CHAPTER CHARACTERIZATION OF SECONDARY METABOLITES IN DURIAN SEEDS AND IN THE FUNGUS-STRESSED DURIAN SEEDS 6.1 Introduction 6.1.1 117 118 Proanthocyanidins in Plants – the Next Milestone in Flavonoid Research 118 6.1.2 Secondary Metabolites in Durian Seeds 122 6.1.3 Objectives 124 6.2 Materials and Methods 124 6.2.1 Materials and Instruments 124 6.2.2 Sample Preparation and Extraction 125 6.2.3 Solvent Extraction and Fractionation of Durian Seeds 126 6.2.4 Extraction and Purification of Oligomeric Proanthocyanidins from Durian Seeds 127 6.2.5 Oligomeric Proanthocyanidins Thiolysis and Identification 129 6.2.6 Quantitative Analysis using Normal Phase HPLC 130 6.2.7 Germination of Durian Seed with (without) Fungal Inoculation 130 VII 6.3 Results and Discussion 132 6.3.1 Chromatographic Fractionation of Durian Seed Extracts 132 6.3.2 Extraction and Structural Elucidation of OPCs 133 6.3.3 Determination of the Degree of Polymerization Procyanidins by Thiolysis 6.3.4 139 Quantification of OPCs in Durian Seed using Normal Phase HPLC 141 6.3.5 Fungal Effect on the Germination of Durian Seeds 6.4 Conclusion CHAPTER CONCLUSIONS AND RECOMMENDATIONS 142 144 147 References 150 APPENDIX 172 VIII SUMMARY Secondary metabolites in plants have attracted worldwide attention partially due to their far reaching health benefits Among the complicated secondary metabolites, phytoalexins are a special group of metabolites that are generated when the plants are under oxidative stress A few studies have reported the bioactivities of phytoalexins but only little research had focused on their potent applications for pharmaceutical and medicinal development The objective of this research was to study the phytoalexin production in stressed plant seeds and to develop phytoalexin enriched food In Chapter and Chapter 2, the backgrounds of phytoalexins generation were introduced, the objectives of the study were proposed In Chapter 3, it was discovered that fungus-stressed germination of black soybeans seeds led to the generation of a group of oxylipins, oxooctadecadienoic acids (KODEs, including 13 - Z, E - KODE, 13 - E, E - KODE, - E, Z - KODE, and - E, E - KODE), and their respective glyceryl esters in addition to glyceollins, a known group of phytoalexins present in wild and fungi infected soybeans The efficiency of four selected fungi in inducing the synthesis of these compounds during black soybean germination was also compared In Chapter 4, the effects of R oligosporus - caused oxidative stress to the germinating black soybeans were further studied Tocopherols, lipid peroxide concentrations, isoflavones, the total phenolics contents and ORAC (Oxygen Radical IX Absorbance Capacity) values in the hydrophilic and lipophilic extracts of the treated black soybeans were studied Results suggested that fungal stress has no significant effects on the antioxidant capacity of the black soybeans In Chapter 5, the fungi-stressed and germinated black soybeans were further processed for functional food development The treated black soybeans were homogenized and fermented with lactic acid bacteria (LAB) to produce soy yogurt The resulting soy yogurt contained a maximum viable cell count of 2.1×108 CFU/mL and had significantly altered the micronutrient profiles resulting in the markedly reduced oligosaccharides but enriched glyceollins, which are known to have anti-cancer properties In Chapter 6, durian seeds were chose for studying the contents of secondary metabolites and phytoalexins generation after germination and fungus-stress Structural identification exhibited the distinctive characteristic of oligomeric proanthocyanidins (OPCs) in durian seeds The yield was 1.8 mg/g of dry seed 13 C and 1H NMR signals showed the presence of procyanidins in the durian seeds The OPCs from durian seeds contain a significant amount of high order B-type oligomers with predominantly epicatechins as the monomeric unit The mean degree of polymerization was determined to be 7.30 The effects of food grade R oligosporus stress on germinating durian seeds were also studied New compounds but with low concentrations were detected by HPLC analysis and were suggested to be phytoalexins synthesized under stress conditions X from the virus of curly top Phytopathoglogy 1940, 30, 673-679 225) Walling， L The myriad plant responses to herbivores J Plant Growth Regul L 2000, 19, 195-216 226) Wang, D Treatment on 38 cases of leucopenia with Xuesu Tang Traditional Chin Med Res 1992, 5, 35-36 227) Wang, X.; Quinn, P Vitamin E and its function in membranes Prog Lipid Res 1999, 38, 309-336 228) Wang, Y C.; Yu, R C.; Yang, H Y.; Chou, C C Sugar and acid contents in soymilk fermented with lactic acid bacteria along or simultaneously with bifidobacteria Food Microbiol 2003, 20, 333-338 229) Wardencki, W.; Michulec, M.; Curylo, J A review of theoretical and practical aspects of solid-phase microextraction in food analysis Int J Food Sci and Tech 2004, 39, 703-717 230) Ware, G W The pesticide book 3rd ed Fresno, CA: Thomson, 1989 231) Watanebe, J.; Kawabata, J.; Kasai, T 9-Oxooctadeca-10, 12-dienoic acids as acetyl-CoA carboxylase inhibitors from red pepper (Capsicum annuum L.) 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Shi, J.; Hession, A O.; Maxwell, C A.; McGonigle, B.; Odell, J T Metabolic engineering to increase isoflavone biosynthesis in soybean seed Phytochem 2003, 63, 753-763 245) Zhu, D.; Hettlarachchy, N S.; Horax R.; Chen P Isoflavone contents in germinated soybean seeds Plant Foods Hum Nutr 2005, 60, 147-151 171 APPENDIX 172 Appendix Table Nutritional Information of Mature Seeds of Soybeans (Glycine max) NDB No: 16108 (unit: /100g) Proximates Water (g) 8.54 Total lipid (fat) (g) 19.94 Energy (kcal) 416 Ash (g) 4.87 Energy (kj) 1742 Carbohydrate, by difference (g) 30.16 Protein (g) 36.49 Fiber, total dietary (g) 9.3 Minerals Calcium, Ca (mg) 277 Sodium, Na(mg) Iron, Fe (mg) 15.7 Zinc, Zn (mg) 4.89 Magnesium, Mg (mg) 280 Copper, Cu (mg) 1.658 Phosphorus, P (mg) 704 Manganese, Mn (mg) 2.517 Potassium, K (mg) 1797 Selenium, Se (mcg) 17.8 Folate, food (mcg) 375 375 Vitamins Vitamin C, total ascorbic acid (mg) Thiamin (mg) 0.874 Folate, DFE (mcg_DFE) Riboflavin (mg) 0.87 Vitamin B-12 (mcg) Niacin (mg) 1.623 Vitamin A, IU (IU) Pantothenic acid (mg) 0.793 Vitamin A, RAE (mcg_RAE) Vitamin B-6 (mg) 0.377 Retinol (mcg) Folate, total (mcg) 375 Vitamin E (alpha-tocopherol) (mg) 0.85 Vitamin K (phylloquinone) (mcg) 47 Folic acid (mcg) Lipids Fatty acids, total saturated (g) 2.884 18:1 undifferentiated (g) 4.348 14:00 (g) 0.055 Fatty acids, total polyunsaturated (g) 11.26 16:00 (g) 2.116 18:2 undifferentiated (g) 9.925 18:00 (g) 0.712 18:3 undifferentiated (g) 1.33 Fatty acids, total monounsaturated (g) 4.404 Cholestero l(mg) 16:1 undifferentiated (g) 0.055 Phytosterols (mg) 161 Tryptophan (g) 0.53 Valine (g) 1.821 Threonine (g) 1.585 Arginine (g) 2.831 Isoleucine (g) 1.77 Histidine (g) 0.984 Leucine (g) 2.972 Alanine (g) 1.719 Lysine (g) 2.429 Aspartic acid (g) 4.589 Methionine (g) 0.492 Glutamic acid (g) 7.068 Cystine (g) 0.588 Glycine (g) 1.687 Phenylalanine (g) 1.905 Proline (g) 2.135 Tyrosine (g) 1.38 Serine (g) 2.115 Amino acids (Adapted from USDA National Nutrient Database for Standard Reference, http://www.nal.usda.gov/fnic/foodcomp/search/ , released 2006) 173 Appendix Table The Nutrional Information of Soymilk and Plain Whole Milk Yogurt (units: /100g) Soymilk, original and Plain whole Nutrient Unit vanilla, milk yogurt unfortified (/100g) (/100g) Proximates Water g 88.05 87.9 Energy kcal 54 61 Energy kj 226 257 Protein g 3.27 3.47 Total lipid (fat) g 1.75 3.25 Ash g 0.65 0.72 Carbohydrate, by difference g 6.28 4.66 Fiber, total dietary g 0.6 Sugars, total g 3.99 4.66 Calcium, Ca mg 25 121 Iron, Fe mg 0.64 0.05 Magnesium, Mg mg 25 12 Phosphorus, P mg 52 95 Potassium, K mg 118 155 Sodium, Na mg 51 46 Zinc, Zn mg 0.12 0.59 Copper, Cu mg 0.128 0.009 Manganese, Mn mg 0.223 0.004 Fluoride, F mg Selenium, Se mcg 4.8 2.2 mg 0.5 Thiamin mg 0.06 0.029 Riboflavin mg 0.069 0.142 Niacin mg 0.513 0.075 Pantothenic acid mg 0.373 0.389 Vitamin B-6 mg 0.077 0.032 Folate, total mcg 18 Folic acid mcg 0 Folate, food mcg 18 Folate, DFE mcg_DFE 18 Choline, total mg 23.6 15.2 Betaine mg 0.8 Vitamin B-12 mcg Minerals 12 Vitamins Vitamin C, total ascorbic acid 0.37 174 Vitamin A, IU IU 99 mcg_RAE 27 Retinol mcg 27 Vitamin E mg 0.11 0.06 IU mcg 0.2 Fatty acids, total saturated g 0.205 2.096 4:00 g 0.096 6:00 g 0.066 8:00 g 0.042 10:00 g 0.093 12:00 g 0.111 14:00 g 0.343 16:00 g 0.15 0.886 18:00 g 0.05 0.317 Fatty acids, total g 0.401 0.893 16:1 undifferentiated g 0.071 17:01 g 0.01 18:1 undifferentiated g 0.31 0.743 20:01 g 0.01 22:1 undifferentiated g 0 Fatty acids, total g 0.961 0.092 18:2 undifferentiated g 0.584 0.065 18:3 undifferentiated g 0.075 0.027 mg 13 Tryptophan g 0.038 0.02 Threonine g 0.108 0.142 Isoleucine g 0.114 0.189 Leucine g 0.186 0.35 Lysine g 0.131 0.311 Methionine g 0.027 0.102 Cystine g 0.032 Phenylalanine g 0.113 0.189 Tyrosine g 0.089 0.175 Vitamin A, RAE (alpha-tocopherol) Vitamin D Vitamin K (phylloquinone) Lipids monounsaturated polyunsaturated Cholesterol Amino acids Valine g 0.117 0.287 Arginine g 0.187 0.104 Histidine g 0.061 0.086 Alanine g 0.104 0.148 Aspartic acid g 0.288 0.275 175 Glutamic acid g 0.487 0.679 Glycine g 0.103 0.084 Proline g 0.147 0.411 Serine g 0.14 0.215 mcg Other Carotene, beta (Adapted from USDA National Nutrient Database for Standard Reference, http://www.nal.usda.gov/fnic/foodcomp/search/, released 2007) 176 Appendix Table The Nutritional Information of Raw or Frozen Durian (Durio zibethinus) (Nutrient Values and Weights are for Edible Portion, Refuse: 68% (Shell and seeds (for raw fruit)) Nutrient Units Value per 100 grams Water g 64.99 Energy kcal 147 Energy kj 615 Protein g 1.47 Total lipid (fat) g 5.33 Ash g 1.12 Carbohydrate, by difference g 27.09 Fiber, total dietary g 3.8 Calcium, Ca mg Iron, Fe mg 0.43 Magnesium, Mg mg 30 Phosphorus, P mg 39 Potassium, K mg 436 Sodium, Na mg Zinc, Zn mg 0.28 Copper, Cu mg 0.207 Manganese, Mn mg 0.325 Vitamin C, total ascorbic acid mg 19.7 Thiamin mg 0.374 Riboflavin mg 0.2 Niacin mg 1.074 Pantothenic acid mg 0.23 Vitamin B-6 mg 0.316 Folate, total mcg 36 Folate, food mcg 36 Vitamin B-12 mcg Vitamin A, IU IU 44 mcg_RAE mcg mg Carotene, beta mcg 23 Carotene, alpha mcg Cryptoxanthin, beta mcg Proximates Minerals Vitamins Vitamin A, RAE Retinol Lipids Cholesterol Other (Adapted from USDA National Nutrient Database for Standard Reference, http://www.nal.usda.gov/fnic/foodcomp/search/, released 2007) 177 The primary mass fragments of KODE in positive and negative ion mode Peak II HREIMS: 294.2 Composition: C18H30O3 The secondary MS of KODE fragment at m/z +295 and m/z -293 The tertiary MS of KODE fragment at m/z 249 of fragment m/z -295 1st Part 178 The tertiary MS of KODE fragment at m/z 249 of fragment m/z -295 1st Part The MS/MS/MS/MS of KODE fragment at m/z 113 of fragment m/z 249 of fragment m/z -295 179 The primary mass fragments of KODE glyceryl ester in positive and negative ion mode [M+H]+ [M-H2O+H]+ [M-H]- 180 The secondary MS of KODE glyceryl ester fragments at m/z +369, m/z +351, m/z -367, and m/z -293 - 181 The tertiary MS of the major secondary fragments of KODE glyceryl ester F: c F ull m s2 33 7.00@ 35.0 [ 0.00 00 0.00] 00 80 R el a ti ve 60 Ab un d a 40 nc e 20 14 9.3 93 61 09 21.2 F: Relative Abundance 148 268 20 50 54 00 m /z 236.2 36 8.6 35 60 97.1 40 185.3 175.3 150 249.1 221.3 193.1 20 293.3 278.2 250.1 219.9 200 310.7 250 300 350 400 m/z c Full ms 319.00@35.00 [ 85.00 1000.00] 100 319.3 F: -c Secondary MS of 319.00@35.00 e 80 60 291.3 40 304.2 20 109.4 147.0 157.5 100 100 209.7 185.5 150 @ 228.3 200 [ 275.3 249.3 264.2 319.9 250 300 350 m/z ] 220.3 F: -c Secondary MS of 220.00@35.00 e 80 60 40 205.2 20 130.2 60 80 100 120 148.2 140 177.5 192.1 160 180 200 F: - c Full ms 131.00@35.00 [ 50.00-1000.00] 100 Relative Abundance 33 7.3 22 2 95 2 75.3 80 100 Relative Abundance 21 5.2 229 F: -c Secondary MS of 293.00@40.00 e 113.4 131.0 Relative Abundance 26 9.2 187 17 5.3 10 50 c Full ms 293.00@40.00 [ 80.00 1000.00] 100 F: 19 F: -c Secondary MS of 337.00@35.00 e 80 213.9 221.0 235.8 220 m/z 240 290.7 260 280 113.1 F: -c Secondary MS of 131.00@35.00 e 60 40 114.1 20 75.4 60 80 85.1 100.9 112.4 100 120 140 160 m/z 180 200 Appendix Figure The primary, secondary and tertiary MS of KODES and KODES glyceryl esters The heated capillary and spray voltage were maintained at 250 °C and 4.5 kV, respectively 182 m, 1.42 m,1.23-1.31 t,0.87 m,6.15 m,7.11 O m,1.23-1.31 m,1.6 m,1.6 O H3 C OH m,2.16 m,6.15 d,6.05 m,1.23-1.31 7.5 7.0 6.5 6.0 t,2.51 m,1.23-1.31 t,2.32 m,1.23-1.31 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 1.0 0.5 (ppm) m,1.23-1.31 m, 1.6 t,0.87 O m,7.11 m,6.14 m,1.41 m,1.23-1.31 m,1.6 O dd,4.12, 4.17 H 3C O m,1.23-1.31 7.5 7.0 t,2.51 d,6.06 6.5 6.0 5.5 m,6.14 m,2.15 5.0 m,1.23-1.31 t,2.32 m,1.23-1.31 4.5 4.0 3.5 3.0 dd,3.57, 3.67 OH OH 2.5 m,3.90 2.0 1.5 (ppm) Appendix Figure A representative 1HNMR spectrum of KODE and KODE glyceryl ester Sample was dissolved in deuterate chloroform and the data were collected at room temperature with operating frequency of 75 MHz 183 Appendix Figure HPLC chromatogram of tocopherols in control (UG) non-stressed germinating black soybeans (G) and R oligosporus stressed germinating black soybeans (GS) with a 3-day time course study 184 ... and developing functional food thereof 1.2 Objectives The overall objective of this research is to study the effects of fungal stress on plant seeds and further study the stressed seeds for novel. .. the changes of nutritional profiles of the fungal stressed bean seeds are also evaluated Based on the investigations, the fungal stressed bean seeds were utilized for developing a novel soy yogurt... especially on the antioxidant capacity of black soybean seeds (Chapter 4) 3) To study the effects of fungal stress on the nutritional value of black soybean seeds and to process a novel black