DEVELOPING PURPLE WHEAT AS A SOURCE OF ANTHOCYANIN

DEVELOPING PURPLE WHEAT AS A SOURCE OF ANTHOCYANIN PIGMENTS FOR FOOD AND NON-FOOD El-Sayed APPLICATIONS M. Abdel-Aal 1 Guelph Food Research Centre, Guelph, Ontario, Canada Euro Food June 2015 1

OUTLINE • Development of colorful wheat and food • Anthocyanin in purple wheat • Role of anthocyanin in life • Purple wheat as a source of anthocyanins • Antioxidant properties of anthocyanins • Preparation for human studies • Conclusions 2

DEVELOPMENT OF COLORFUL WHEAT AND PURPLE WHEAT FOODS 3

Abdel-Aal and Hucl, Cereal Chemistry, 1999, 76, 350 4

0. 8 Cy-3 --Glu BW wholemeal BW bran PW wholemeal PW bran Blue barley Absorbance 0. 6 0. 4 0. 2 0. 0 440 480 520 560 600 Wavelength (nm) Absorbance spectra of Cy-3 -Glu and wheat extracts Abdel-Aal and Hucl, Cereal Chemistry, 1999, 76, 350 5

Purple wheat Red wheat 6 Blue wheat

CDC Primepurple wheat 2013 Antho. Grain. TM 7

Wheat Milling Fractions Pericarp (5 -6%) Testa (3 -4%) Aleurone layer (7 -8%) Endosperm (81 -84%) Embryo (2 -3%) Wheat kernel Yield, % 7. 1 ACNs, mg/kg 818 22. 6 66. 8 100 495 32 212 Abdel-Aal et al. , J. Agric. Food Chem. , 2006, 54, 4696 8

Wheat Flour Uniqueness Gliadin >30 gliadins Glutenin 4 -6 HMW & 15 LMW 50 protein subunits 9 Gluten

Purple Wheat Food Applications • Breads • Breakfast Cereals • Snacks • Noodles • Crackers • Pancakes 10

Purple Wheat Foods Pancake Flakes Bread Noodles 11 Muffin

ANTHOCYANINS IN PURPLE WHEAT 12

Anthocyanin Structures R 1 3' 2' 8 HO 7 A 5 1' O 1 2 C 6 4 B 3 6' O Delphinidin-3 -glucoside: R 1=OH, R 2=OH 4' OH 5' R 2 Cyanidin-3 -glucoside: R 1=OH, R 2=H Petunidin-3 -glucoside: R 1=OCH 3, R 2=OH Pelargonidin-3 -glucoside: R 1=H, R 2=H glucose OH Peonidin-3 -glucoside: R 1=OCH 3, R 2=H Malvidin-3 -glucoside: R 1=OCH 3, R 2=OCH 3 Organic acid (aliphatic or phenolic) 13

Characterization of anthocyanin compounds in purple wheat bran 14

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ROLE OF ANTHOCYANINS IN LIFE 17

Plants Ø Pollination and seed dispersal Ø Protection against UV Ø Reduction of oxidative stress 18

Human Health Ø Biological antioxidant Ø Anti-cancer Ø Anti-inflammatory effect Ø Anti-diabetic effect Ø Ocular effect 19

Food Industry Ø Natural colorant Ø Dietary antioxidant Ø Functional ingredient 20

Cosmetic and healthcare Industry Ø Natural colorants Ø Natural antioxidants 21

Anthocyanin Consumption Daily intake: 12. 5 mg of the total intake: 45% cyanidin 21% delphinidin 77% nonacylated 73% monoglucoside Wu et al. , J. Agric. Food Chem. , 2006, 54, 4069. 22

PURPLE WHEAT AS A SOURCE OF ANTHOCYANINS 23

Purple Wheat Kernels Abrasive or Roller Milling White endosperm Bran Aqueous ethanol extraction Bakery products Column chromatography Purification (amberlite XAD-7) Drying Solvent proof oven Anthocyanin powder Characterization Composition, color, stability antioxidant and biological properties 24

Purification by column chromatography 25

Average yield anthocyanin concentration in isolates Batch size Yield (g/batch) Purified-Amberlite 10 g 0. 21 50 g 0. 79 100 g 2. 05 Purified-C 18 50 g 0. 75 Non-purified 50 g 1. 17 Yield (mg/g) Concentration of Concentration anthocyanin factora (mg/g) 21. 0 15. 8 20. 5 57. 4 38. 9 34. 7 135 91 81 15. 0 33. 9 80 23. 4 13. 7 32 a. Ratio of anthocyanin concentration in powder to that in purple wheat bran. 26

ANTIOXIDANT PROPERTIES OF ANTHOCYANINS 27

DPPH scavenging capacity of blue wheat powder μmole/g Product bran and μmole/g 10 min 30 min 46 2526 40 Bran 1686 Anthocyanin powder Abdel-Aal et al. , J Agric. Food Chem. , 2008, 56, 11171 28

ABTS scavenging capacity of blue wheat powder μmole/g Product bran and μmole/g 3 min 10 min 57 23 Bran 2925 1610 Anthocyanin powder Abdel-Aal et al. , J Agric. Food Chem. , 2008, 56, 11171 29

Inhibition of human LDL cholesterol oxidation (μmole diene/g LDL/mg sample) Product 2 hrs 8 hrs 12 hrs 24 hrs Control (no 381 528 530 antioxidant) 1 17 32 Bran 0. 055 0. 062 0. 29 Anthocyanin powder Abdel-Aal et al. , J Agric. Food Chem. , 2008, 56, 11171 30 584 33 1. 44

DPPH scavenging capacity of anthocyanins Anthocyani μmole/g n 10 min 30 min Dp-3 -Glu Cy-3 -Glu Dp-3 -Rut Cy-3 -Rut 792 1113 567 3967 831 1232 613 4346 Abdel-Aal et al. , J Agric. Food Chem. , 2008, 56, 11171 31

ABTS scavenging capacity of anthocyanins Anthocyanin μmole/g Dp-3 -Glu Cy-3 -Glu Dp-3 -Rut Cy-3 -Rut 3 min 10 min 192 533 232 1673 382 1015 430 3261 Abdel-Aal et al. , J Agric. Food Chem. , 2008, 56, 11171 32

Inhibition of human LDL cholesterol oxidation (μmole diene/g LDL/mg sample) Compound 2 hrs 8 hrs 12 hrs 24 hrs Control Dp-3 -Glu Cy-3 -Glu Dp-3 -Rut Cy-3 -Rut BHT 528 0. 119 0. 695 0. 092 3. 130 23. 0 381 0. 042 0. 308 0. 047 1. 251 19. 4 530 0. 263 0. 720 0. 272 3. 196 30. 5 584 0. 244 0. 928 0. 267 4. 018 36. 6 Abdel-Aal et al. , J Agric. Food Chem. , 2008, 56, 11171 33

HUMAN STUDIES 34

Products Anthocyanin content of Purple Wheat anthocyanin-enriched products measured by spectrophotometric Anthocyanin (µg/g) method Fresh Dry matter weight basis Wholegrain bread 65 101 Wholegrain muffin 49 78 Wholegrain pancake 96 167 Wholegrain flakes (uncooked) 120 135 Wholegrain flakes (cooked) 48 116 Wholegrain instant flakes (uncooked) 129 146 Wholegrain instant flakes (cooked) 30 116 35

Nutrient content of Purple Wheat anthocyaninenriched Products (dry basis) Product s Wholegrain bread + bran Wholegrain muffin Wholegrain flakes (uncooked) Wholegrain flakes (cooked) Wholegrain instant flakes (uncooked) Wholegrain instant flakes (cooked) Wholegrain pancake Wholegrain flour Protein Starch Fiber Fat 18. 0 16. 5 9. 2 16. 2 17. 0 17. 5 18. 8 16. 8 11. 5 56. 8 55. 4 19. 6 60. 6 57. 6 55. 4 61. 2 36. 0 56. 4 11. 9 16. 4 18. 1 12. 6 14. 0 12. 7 13. 6 20. 7 1. 8 6. 4 5. 1 14. 5 2. 1 1. 5 1. 7 1. 3 7. 9 12. 6 36

1 st study: bioavailability of anthocyanins and phenolic & plasma antioxidant capacity Foods: Purple wheat wholegrain bread Purple wheat flakes Study participants: Healthy individuals (18 -55 Yrs, BMI 18 -25) Study design: Randomized crossover acute meal study 37

2 nd study: intervention study, oxidative stress and inflammation responses Foods: Purple wheat wholegrain bread Purple wheat flakes Study participants: Individuals at risk Study design: Randomized placebo crossover acute meal study 38

Take Home Messages Ø Purple wheat holds a promise for the development of natural colorants and/or natural antioxidants Ø Purple wheat can be processed into a variety of staple foods for healthy diets Ø Dry and wet separation technologies can be used to isolate anthocyanins from purple wheat Ø More research is underway to evaluate bioavailability and health benefits of purple wheat products 39

Acknowledgments Collaborators • Dr. Pierre Hucl, U of S • Dr. Amanda Wright, U of G • Mr. Mark Pickard, Saskatoon Research technicians • Iwona Rabalski • Marta Hernandez Students Partners & Grants • Tamer Gamel, U of G • Infra. Ready Inc, Saskatoon • Tyler O’Leary, U of G • Canadian Food Innovation Cluster • Riley De. Forest, U of G • Agriculture Development Fund of Saskatchewan • Jacklyn Shipp, U of G • Agriculture and Agri-Food Canada • Jessica Simons 40

THANK YOU Questions AAFC Vision: Driving innovation and ingenuity to build a world leading agricultural and food economy for the benefit of all Canadians AAFC Mission: to provide leadership in the growth and development of a competitive, innovative and sustainable Canadian agriculture and agri-food sector 41