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DEVELOPING DEVELOPING PURPLE WHEAT PURPLE WHEAT AS A SOURCE AS A SOURCE OFOF

ANTHOCYANIN PIGMENTS FOR FOOD ANDANTHOCYANIN PIGMENTS FOR FOOD ANDNON-FOOD APPLICATIONSNON-FOOD APPLICATIONS

El-Sayed M. Abdel-AalEl-Sayed M. Abdel-Aal11Guelph Food Research Centre, Guelph, Ontario, CanadaGuelph Food Research Centre, Guelph, Ontario, Canada

Euro Food June 2015Euro Food June 2015

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OUTLINEOUTLINE

• Development of colorful wheat and foodDevelopment of colorful wheat and food

• Anthocyanin in purple wheatAnthocyanin in purple wheat

• Role of anthocyanin in lifeRole of anthocyanin in life

• Purple wheat as a source of anthocyaninsPurple wheat as a source of anthocyanins

• Antioxidant properties of anthocyaninsAntioxidant properties of anthocyanins

• Preparation for human studiesPreparation for human studies

• ConclusionsConclusions

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DEVELOPMENT OF COLORFUL WHEATDEVELOPMENT OF COLORFUL WHEATAND PURPLE WHEAT FOODSAND PURPLE WHEAT FOODS

4

Anthocyanin (mg/kg)

0 100 200 300 400 500 600

Dis

trib

uti

on

nu

mb

er

0

10

20

30

40

50

Range = 34.6-507.2Mean = 183.0SD = 95.7SE = 7.6n = 160

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

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Absorbance spectra of Cy-3-Glu and wheat extracts

Wavelength (nm)

440 480 520 560 600

Ab

so

rba

nc

e

0.0

0.2

0.4

0.6

0.8

Cy-3--Glu BW wholemeal BW bran PW wholemeal PW bran Blue barley

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

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Blue wheat Red wheat Purple wheat

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CDC Primepurple wheat2013

AnthoGrainTM

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Wheat Milling FractionsWheat Milling Fractions

Testa (3-4%)

Aleurone layer (7-8%)

Embryo (2-3%)

Pericarp (5-6%)

Endosperm (81-84%)

81849532212

ACNs, mg/kg

7.122.666.8100

Yield, %

Abdel-Aal et al., J. Agric. Food Chem., 2006, 54, 4696 Wheat kernelWheat kernel

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GlutenGlutenGliadinGliadin GluteninGlutenin

50 protein subunits50 protein subunits

Wheat Flour UniquenessWheat Flour Uniqueness

4-6 HMW & 15 LMW 4-6 HMW & 15 LMW >30 gliadins>30 gliadins

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Purple Wheat Food ApplicationsPurple Wheat Food Applications

• Breakfast CerealsBreakfast Cereals

• BreadsBreads

• SnacksSnacks

• CrackersCrackers

• NoodlesNoodles

• PancakesPancakes

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Purple Wheat FoodsPurple Wheat Foods

BreadBreadPancakePancake

NoodlesNoodlesFlakesFlakes MuffinMuffin

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ANTHOCYANINS IN PURPLE WHEATANTHOCYANINS IN PURPLE WHEAT

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OH

1

35

6

8

7

1'

2'

3'4'

6'

5'O

OH

HO

R1

R2

O glucose

2

4

Pelargonidin-3-glucoside: R1=H, R2=H

Cyanidin-3-glucoside: R1=OH, R2=H

Malvidin-3-glucoside: R1=OCH3, R2=OCH3

Peonidin-3-glucoside: R1=OCH3, R2=H

Petunidin-3-glucoside: R1=OCH3, R2=OH

Delphinidin-3-glucoside: R1=OH, R2=OH

Anthocyanin StructuresAnthocyanin Structures

Organic acid (aliphatic or phenolic)

A

B

C

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Compound Anthocyanin # of isomeric compounds

Major ion pair Concentration of anthocyanin

(µg/g) 1 Cy-diGlu 9 611/287 1.67 2 Dp-3-Glu 1 465/303 0.09 3 Dp-3-Rut 4 611/303 3.67 4 Dp-MalGlu 5 551/303 3.54 5 Dp-SucGlu 3 565/303 tr 6 Cy-3-Glu 1 449/287 116.66 7 Cy-3-Rut 1 595/287 85.06 8 Cy-MalGlu 4 535/287 150.28 9 Cy-SucGlu 3 549/287 1.00 10 Cy-MalSucGlu 1 635/287 tr 11 Pt-3-Glu 2 479/317 0.19 12 Pt-3-Rut 1 625/317 0.05 13 Pt-MalGlu 1 565/317 3.23 14 Pg-3-Glu 1 433/271 0.91 15 Pg-Rut 3 579/271 0.35 16 Pg- MalGlu 3 519/271 1.17 17 Pg-SucGlu 3 533/271 0.19 18 Pn-3-Glu 4 463/301 27.37 19 Pn-Rut 3 609/301 7.38 20 Pn-MalGlu 6 549/301 18.38 21 Pn-Glu Succ 3 563/301 0.03 22 Mv 3 Glu 3 493/331 0.28 23 Mv Rut 2 639/331 2.55 24 Mv Glu Mal 4 579/331 0.53 25 Mv Glu Succ 3 593/331 1.28 26 Mv Glu Mal Succ 1 679/331 0.16 Total 75 - 426.02

Characterization of anthocyanin compounds in purple wheat branCharacterization of anthocyanin compounds in purple wheat bran

15

16

0100

Cy-3-Gl(+ ve)

449

287

465

303Dp-3-Gl

(+ ve)

0

100

100 1000

Pn-3-Gl(+ ve)

463

301

100 1000

Pt-3-Gl(+ ve)

479

317

100 1000100 1000

100

1000

Cy-dGl(+ ve)

611

287

100 1000

Cy-SGl(+ ve)287

549

100 1000

100

1400

Cy-3-Gl(- ve)

0

284

447

493 659

1343

895

M/Z

Ab

und

ance

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ROLE OF ANTHOCYANINS IN LIFEROLE OF ANTHOCYANINS IN LIFE

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Protection against UVProtection against UV

Pollination and seed dispersalPollination and seed dispersal

Reduction of oxidative stressReduction of oxidative stress

PlantsPlants

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Human HealthHuman Health

Biological antioxidantBiological antioxidant

Anti-inflammatory effectAnti-inflammatory effect

Anti-cancerAnti-cancer

Anti-diabetic effectAnti-diabetic effect

Ocular effectOcular effect

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Natural colorantNatural colorant

Dietary antioxidantDietary antioxidant

Food IndustryFood Industry

Functional ingredient Functional ingredient

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Cosmetic and healthcare IndustryCosmetic and healthcare Industry

Natural colorantsNatural colorants

Natural antioxidantsNatural antioxidants

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Daily intake: 12.5 mgDaily intake: 12.5 mg

of the total intake:of the total intake:45% cyanidin45% cyanidin

21% delphinidin21% delphinidin77% nonacylated77% nonacylated

73% monoglucoside73% monoglucoside

Wu et al., J. Agric. Food Chem., 2006, 54, 4069.

Anthocyanin ConsumptionAnthocyanin Consumption

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PURPLE WHEAT AS A SOURCE OFPURPLE WHEAT AS A SOURCE OFANTHOCYANINSANTHOCYANINS

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PPurple urple WWheat heat KKernelsernels

Abrasive or Roller MillingAbrasive or Roller Milling

White endospermWhite endosperm

Aqueous ethanol extractionAqueous ethanol extraction

PurificationPurification

DryingDrying

Column chromatographyColumn chromatography(amberlite XAD-7)(amberlite XAD-7)

BranBran

Anthocyanin powderAnthocyanin powder

CharacterizationCharacterization

Solvent proof ovenSolvent proof oven

Composition, color, stabilityComposition, color, stabilityantioxidant and biologicalantioxidant and biological

propertiesproperties

Bakery productsBakery products

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Purification by column chromatographyPurification by column chromatography

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Batch size Yield(g/batch)

Yield(mg/g)

Concentration of anthocyanin (mg/g)

Concentration factora

Purified-Amberlite10g 0.21 21.0 57.4 13550g 0.79 15.8 38.9 91100g 2.05 20.5 34.7 81Purified-C1850g 0.75 15.0 33.9 80Non-purified50g 1.17 23.4 13.7 32

Average yield and anthocyanin concentration in isolates

aRatio of anthocyanin concentration in powder to that in purple wheat bran.

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ANTIOXIDANT PROPERTIESANTIOXIDANT PROPERTIESOF ANTHOCYANINSOF ANTHOCYANINS

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DPPH scavenging capacity of blue wheatDPPH scavenging capacity of blue wheatbran and powderbran and powder

BranAnthocyanin powder

Product

401686

μmole/g10 min

462526

μmole/g30 min

Abdel-Aal et al., J Agric. Food Chem., 2008, 56, 11171

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ABTS scavenging capacity of blue ABTS scavenging capacity of blue wheatwheat

bran and powderbran and powder

BranAnthocyanin powder

Product

231610

μmole/g3 min

572925

μmole/g10 min

Abdel-Aal et al., J Agric. Food Chem., 2008, 56, 11171

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Inhibition of human LDL cholesterol oxidationInhibition of human LDL cholesterol oxidation((μμmole diene/g LDL/mg sample)mole diene/g LDL/mg sample)

Control (no antioxidant)BranAnthocyanin powder

Product

52817

0.062

8 hrs

53032

0.29

12 hrs

3811

0.055

2 hrs

58433

1.44

24 hrs

Abdel-Aal et al., J Agric. Food Chem., 2008, 56, 11171

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DPPH scavenging capacity of anthocyaninsDPPH scavenging capacity of anthocyanins

Dp-3-GluCy-3-GluDp-3-RutCy-3-Rut

Anthocyanin

7921113567

3967

μmole/g10 min

8311232613

4346

μmole/g30 min

Abdel-Aal et al., J Agric. Food Chem., 2008, 56, 11171

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ABTS scavenging capacity of anthocyaninsABTS scavenging capacity of anthocyanins

Dp-3-GluCy-3-GluDp-3-RutCy-3-Rut

Anthocyanin

1925332321673

μmole/g3 min

38210154303261

μmole/g10 min

Abdel-Aal et al., J Agric. Food Chem., 2008, 56, 11171

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Inhibition of human LDL cholesterol oxidationInhibition of human LDL cholesterol oxidation((μμmole diene/g LDL/mg sample)mole diene/g LDL/mg sample)

ControlDp-3-GluCy-3-GluDp-3-RutCy-3-RutBHT

Compound

5280.1190.6950.0923.13023.0

8 hrs

5300.2630.7200.2723.19630.5

12 hrs

3810.0420.3080.0471.25119.4

2 hrs

5840.2440.9280.2674.01836.6

24 hrs

Abdel-Aal et al., J Agric. Food Chem., 2008, 56, 11171

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HUMAN STUDIESHUMAN STUDIES

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Products Anthocyanin (µg/g)

Fresh weight basis

Dry matterbasis

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

Anthocyanin content of Purple Wheat anthocyanin-enrichedproducts measured by spectrophotometric method

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Nutrient content of Purple Wheat anthocyanin-enrichedProducts (dry basis)

18.016.59.216.217.017.518.816.811.5

Wholegrain breadWholegrain bread + branWholegrain muffinWholegrain flakes (uncooked)Wholegrain flakes (cooked)Wholegrain instant flakes (uncooked)Wholegrain instant flakes (cooked)Wholegrain pancakeWholegrain flour

Products Protein

56.855.419.660.657.655.461.236.056.4

Starch

11.916.418.112.614.012.713.620.71.8

Fiber

6.45.114.52.11.51.71.37.912.6

Fat

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11stst study: bioavailability of anthocyanins and phenolic acids study: bioavailability of anthocyanins and phenolic acids & plasma antioxidant capacity& plasma antioxidant capacity

Foods:Foods:Purple wheat wholegrain breadPurple wheat wholegrain breadPurple wheat flakesPurple wheat flakes

Study participants:Study participants:Healthy individuals (18-55 Yrs, BMI 18-25)Healthy individuals (18-55 Yrs, BMI 18-25)

Study design:Study design:Randomized crossover acute meal study Randomized crossover acute meal study

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22ndnd study: intervention study, oxidative stress study: intervention study, oxidative stress and inflammation responsesand inflammation responses

Foods:Foods:Purple wheat wholegrain breadPurple wheat wholegrain breadPurple wheat flakesPurple wheat flakes

Study participants:Study participants:Individuals at riskIndividuals at risk

Study design:Study design:Randomized placebo crossover acute meal study Randomized placebo crossover acute meal study

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Dry and wet separation technologies can be used to isolateDry and wet separation technologies can be used to isolate anthocyanins from purple wheatanthocyanins from purple wheat

Take Home MessagesTake Home Messages

Purple wheat holds a promise for the development of natural Purple wheat holds a promise for the development of natural colorants and/or natural antioxidantscolorants and/or natural antioxidants

Purple wheat can be processed into a variety of staple foodsPurple wheat can be processed into a variety of staple foods for healthy dietsfor healthy diets

More research is underway to evaluate bioavailability and healthMore research is underway to evaluate bioavailability and health benefits of purple wheat productsbenefits of purple wheat products

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AcknowledgmentsAcknowledgments

CollaboratorsCollaborators• Dr. Pierre Hucl, U of SDr. Pierre Hucl, U of S• Dr. Amanda Wright, U of GDr. Amanda Wright, U of G• Mr. Mark Pickard, SaskatoonMr. Mark Pickard, Saskatoon

StudentsStudents• Tamer Gamel, U of GTamer Gamel, U of G• Tyler O’Leary, U of GTyler O’Leary, U of G• Riley DeForest, U of GRiley DeForest, U of G• Jacklyn Shipp, U of GJacklyn Shipp, U of G• Jessica SimonsJessica Simons

Research techniciansResearch technicians• Iwona RabalskiIwona Rabalski• Marta HernandezMarta Hernandez

Partners & GrantsPartners & Grants• InfraReady Inc, SaskatoonInfraReady Inc, Saskatoon• Canadian Food Innovation ClusterCanadian Food Innovation Cluster• Agriculture Development Fund of SaskatchewanAgriculture Development Fund of Saskatchewan• Agriculture and Agri-Food CanadaAgriculture and Agri-Food Canada

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THANK YOUTHANK YOUQuestions

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