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

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

3. 3 DEVELOPMENT OF COLORFUL WHEAT AND PURPLE WHEAT FOODS

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

5. 5 Absorbance spectra of Cy-3-Glu and wheat extracts Abdel-Aal and Hucl, Cereal Chemistry, 1999, 76, 350

6. 6 Blue wheat Red wheatPurple wheat

7. 7 CDC Primepurple wheat 2013 AnthoGrain TM

8. 8 Wheat Milling Fractions Testa (3-4%) Aleurone layer (7-8%) Embryo (2-3%) Pericarp (5-6%) Endosperm (81-84%) 818 495 32 212 ACNs, mg/kg 7.1 22.6 66.8 100 Yield, % Abdel-Aal et al., J. Agric. Food Chem., 2006, 54, 4696 Wheat kernel

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

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

11. 11 Purple Wheat Foods BreadPancake Noodles Flakes Muffin

12. 12 ANTHOCYANINS IN PURPLE WHEAT

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

14. 14 Characterization of anthocyanin compounds in purple wheat bran

15. 15

16. 16

17. 17 ROLE OF ANTHOCYANINS IN LIFE

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

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

20. 20  Natural colorant  Dietary antioxidant Food Industry  Functional ingredient

21. 21 Cosmetic and healthcare Industry  Natural colorants  Natural antioxidants

22. 22 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. Anthocyanin Consumption Anthocyanin Consumption

23. 23 PURPLE WHEAT AS A SOURCE OF ANTHOCYANINS

24. 24 P urple W heat K ernels Abrasive or Roller Milling White endosperm Aqueous ethanol extraction Purification Drying Column chromatography (amberlite XAD-7) Bran Anthocyanin powder Characterization Solvent proof oven Composition, color, stability antioxidant and biological properties Bakery products

25. 25 Purification by column chromatography

26. 26 Batch sizeYield (g/batch) Yield (mg/g) Concentration of anthocyanin (mg/g) Concentration factor a Purified-Amberlite 10g 0.2121.0 57.4 135 50g 0.7915.8 38.9 91 100g 2.0520.5 34.7 81 Purified-C18 50g 0.7515.0 33.9 80 Non-purified 50g 1.1723.4 13.7 32 Average yield and anthocyanin concentration in isolates a Ratio of anthocyanin concentration in powder to that in purple wheat bran.

27. 27 ANTIOXIDANT PROPERTIES OF ANTHOCYANINS

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

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

30. 30 Inhibition of human LDL cholesterol oxidation (μmole diene/g LDL/mg sample) Control (no antioxidant) Bran Anthocyanin powder Product 528 17 0.062 8 hrs 530 32 0.29 12 hrs 381 1 0.055 2 hrs 584 33 1.44 24 hrs Abdel-Aal et al., J Agric. Food Chem., 2008, 56, 11171

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

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

33. 33 Inhibition of human LDL cholesterol oxidation (μmole diene/g LDL/mg sample) Control Dp-3-Glu Cy-3-Glu Dp-3-Rut Cy-3-Rut BHT Compound 528 0.119 0.695 0.092 3.130 23.0 8 hrs 530 0.263 0.720 0.272 3.196 30.5 12 hrs 381 0.042 0.308 0.047 1.251 19.4 2 hrs 584 0.244 0.928 0.267 4.018 36.6 24 hrs Abdel-Aal et al., J Agric. Food Chem., 2008, 56, 11171

34. 34 HUMAN STUDIES

35. 35 ProductsAnthocyanin (µg/g) Fresh weight basis Dry matter basis Wholegrain bread 65101 Wholegrain muffin 4978 Wholegrain pancake 96167 Wholegrain flakes (uncooked) 120135 Wholegrain flakes (cooked) 48116 Wholegrain instant flakes (uncooked) 129146 Wholegrain instant flakes (cooked) 30116 Anthocyanin content of Purple Wheat anthocyanin-enriched products measured by spectrophotometric method

36. 36 Nutrient content of Purple Wheat anthocyanin-enriched Products (dry basis) 18.0 16.5 9.2 16.2 17.0 17.5 18.8 16.8 11.5 Wholegrain bread Wholegrain bread + bran Wholegrain muffin Wholegrain flakes (uncooked) Wholegrain flakes (cooked) Wholegrain instant flakes (uncooked) Wholegrain instant flakes (cooked) Wholegrain pancake Wholegrain flour ProductsProtein 56.8 55.4 19.6 60.6 57.6 55.4 61.2 36.0 56.4 Starch 11.9 16.4 18.1 12.6 14.0 12.7 13.6 20.7 1.8 Fiber 6.4 5.1 14.5 2.1 1.5 1.7 1.3 7.9 12.6 Fat

37. 37 1 st study: bioavailability of anthocyanins and phenolic acids & plasma antioxidant capacity & 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

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

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

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

41. 41 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