Enhancing Food Nutritional Quality. Fe I ↑ WtPEMVit. A Estimates of World Wide Malnutrition.

Enhancing Food Enhancing Food Nutritional QualityNutritional Quality

Fe I

↑ Wt

PEM Vit. A

Estimates of World Wide Malnutrition

Micro-Nutrient Deficiency Micro-Nutrient Deficiency Diseases in the Developing Diseases in the Developing

WorldWorld↓ ↓ Vit. A impairs immune system, Vit. A impairs immune system, increasing the risk of illness and death. increasing the risk of illness and death. ↑ blindness.↑ blindness.

↓ ↓ Iodine causes reduced mental Iodine causes reduced mental capacity and irreversible brain damage,capacity and irreversible brain damage,

↓ ↓ Iron results in extreme fatigue with Iron results in extreme fatigue with adult diminished work capacity (15%). adult diminished work capacity (15%). Children are less able to concentrate Children are less able to concentrate and learn. Pregnant women have an ↑ and learn. Pregnant women have an ↑ risk of death during childbirth (20 % in risk of death during childbirth (20 % in Asia and Africa),Asia and Africa),

Chronic Disease in the Chronic Disease in the Developed WorldDeveloped World

Heart disease and Cancer cause Heart disease and Cancer cause most deathsmost deaths

Chronic diseases are associated Chronic diseases are associated with…….. with…….. – Low activity levels – Excess kilocalories– Excess and types of dietary fat– Low micro-nutrients– Low phytochemicals?

"Saber"

A New High Yielding Rice Cultivar for Producers in the

Southern U.S.

Anna McClungToni Marchetti

Christine BergmanRobert Fjellstrom

USDA-ARS

Why?Why?

World Food World Food SupplySupply– FAO (2001)– 2,807

Kcal/person/d– 76 g

protein/person/d (PEM)

How Can How Can WeWe Decrease Decrease Malnutrition?Malnutrition?

Vitamin and Mineral Vitamin and Mineral Supplements Supplements (acute problems) (acute problems)

Food Fortification Food Fortification (reaches only some) (reaches only some)

Biotechnology Biotechnology (sustainable solutions?) (sustainable solutions?)

Traditional, Molecular assisted and Traditional, Molecular assisted and Mutation Plant Breeding Mutation Plant Breeding (sustainable (sustainable solutions)solutions)

Food FortificationFood Fortification Add nutrients to staple foodsAdd nutrients to staple foods Add nutrients to processed foodsAdd nutrients to processed foods Blend more than one staple food Blend more than one staple food

togethertogether

Pasta Fortification

• Excess wheat not suitable for use in bread or pasta

• Wheat is low in lysine

• Wheat + legumes = a complete protein

• (Soft wheat + 30% cowpea) x special processing method = high protein pasta

Thai Poverty and Hunger Reduction Program

(1984)

1. Production of diversified foods for home consumption

2. Skill development and credit for commercial food processing

3. Fortification of Thai instant noodles with vitamin A, iron and iodine

4. Mandatory nutrition labeling of food products

5. Promotion of 9 healthy diets for age-specific groups such as infants and young children, adolescent girls and pregnant women

6. Free or highly subsidized health care

7. Surveillance of children under five years and children in primary school

Progress in Reducing Underweight Children (< five)

Source: Bureau of Health Promotion, Ministry of Public Health, Government of Thailand, 1998

Foods being fortified with……….

Vitamin A Iron

Bolivia Sugar --

Botswana Tsabana --

Brazil* Sugar --

Chile -- Wheat flour

Egypt -- Wheat flour

El Salvador Sugar Wheat flour

Grenada -- Wheat flour

Guatemala Sugar Wheat flour

Honduras Sugar --

India Cow's milk --

Crop Genetic Crop Genetic ModificationModification

Over, under or blocked expression Over, under or blocked expression of native genesof native genes

Insertion of non-native genesInsertion of non-native genes Traits in commercial cultivars today Traits in commercial cultivars today

= herbicide resistance= herbicide resistance

Genetically Modified Crops of Today and Tomorrow

RiceRiceAccounts for Accounts for 80% of the total 80% of the total

kilocalorieskilocalories and up to and up to 20% of the 20% of the proteinprotein consumed by ~2.7 billion consumed by ~2.7 billion Asians, or half the world's population.Asians, or half the world's population.

The most rapidly growing food source The most rapidly growing food source in Africa in Africa

Fortification of wheat flour with iron Fortification of wheat flour with iron etc. has been a successful strategy to etc. has been a successful strategy to decrease deficiencies in developed decrease deficiencies in developed countries. Similar fortification of rice countries. Similar fortification of rice is not as practical.is not as practical.

““Golden Rice”Golden Rice”

• Rice does not contain beta-carotene

• Genes from daffodils and bacteria isoprenoid pathways were cloned

• Rice was transformed with genes that code for:

1. Phytoene synthase

2. Phytoene desaturase

3. Lycopene cyclase

4. Carotene desaturase

• Color due to beta carotene

• Could provide 15 - 100% of daily requirements• Biotechnology can increase crop nutrients and controversy

• Significantly reduce the incidence and severity of vitamin A deficiency?•Bioavailability? Yield drag? Seed cost? Consumer acceptance?

"Golden Rice"

High-Iron Rice?High-Iron Rice? Ferritin gene from Phaseolus = 2.5-fold ↑ in Fe content Cysteine-rich polypeptides enhance iron absorption → a

metallothionin gene from Oryza has been overexpressed = a 7.0-fold increase in endosperm cysteine

A thermotolerant phytase from Aspergillus fumigatus To prevent activity which could interfere with

germination, the enzyme was excreted into the extracellular space

In small intestine simulation experiments the phytase degraded phytate to zero within 1.0 hour

However, in transgenic rice the enzyme did not refold properly after cooking, i.e., it lost it’s thermotolerance

The enzyme has been targeted to the phytase storage vesicles to reduce the phytate content directly. Effect on germination?

"Golden Rice" was possible "Golden Rice" was possible because…..because….. (I. Potrykus) (I. Potrykus)

I had stable, public funding over a long period of time which I could use independent of the opinion of others.

I had with Peter Beyer the perfect partner, who understood the underlying science and provided the necessary genes and analytical expertise.

The Rockefelller Foundation was willing to add substantial financial support over a long time period.

The Swiss Federal Institute of Technology supported the concept of strategic research for developing countries.

The project was embedded in an enthusiastic group of coworkers (over 60), all motivated to contribute to food security with their work.

I was naive enough to believe in it’s success……….

What Do You Think?What Do You Think?

"If anyone tells you that GMOs are going to feed the world, tell them that it is not… To feed the world takes political and financial will – it’s not about production and distribution.“ Steve Smith, Novartis

Plant BreedingPlant BreedingSince the origins of agriculture selection Since the origins of agriculture selection

pressure has created new crops pressure has created new crops Selection is directed evolution.Selection is directed evolution.A plant product (a “phenotype”) is the A plant product (a “phenotype”) is the

result of the plant’s genotype interacting result of the plant’s genotype interacting with the environment. with the environment.

Plant breeders develop varieties Plant breeders develop varieties (“packages of genes) that are specifically (“packages of genes) that are specifically adapted to certain environments, and adapted to certain environments, and which have specific traits that which have specific traits that consumers/users/processors demand. consumers/users/processors demand.

Rice CultivarsRice Cultivars

CH 1007 EAS 3 Romeno

N2404 Taipei 177

Yachikogane

Barbra McClintock (1902 – Barbra McClintock (1902 – 1992)1992)

Nobel Prize Winner (1983)Nobel Prize Winner (1983)"Over the many years, I truly "Over the many years, I truly

enjoyed not being required to defend enjoyed not being required to defend my interpretations. I could just work my interpretations. I could just work with the greatest of pleasure. I never with the greatest of pleasure. I never felt the need nor the desire to felt the need nor the desire to defend my views. If I turned out to defend my views. If I turned out to be wrong, I just forgot that I ever be wrong, I just forgot that I ever held such a view. It didn't matter." --held such a view. It didn't matter." --Barbara McClintock, 1983Barbara McClintock, 1983

OilseedOilseedIn Canada rapeseed was made into a In Canada rapeseed was made into a

new crop: "canola." (Canadian + oil)new crop: "canola." (Canadian + oil) Canola is essentially free of erucic Canola is essentially free of erucic

acid (C 22:1) (linked w/ HRT DZ) and acid (C 22:1) (linked w/ HRT DZ) and glucosinolates glucosinolates – ~ 5% saturated, 60% mono-unsaturated,

30% polyunsaturated lipids– Meal is palatable to livestock

World's 2World's 2ndnd – 3 – 3rdrd most important food most important food oil source oil source

Baldur Rosmund Stefansson, PhD (1917 – 2002): “Father of Canola"

RiceRice

White versus White versus brown? brown? Why?Why?

Rancidity = TG → Rancidity = TG → Free Free FA → FA →

aldehydes + aldehydes + ketonesketones40 M metric T/year40 M metric T/year Source of oil, Source of oil,

vitamins, minerals, vitamins, minerals, dietary fiber, dietary fiber, protein, protein, phytochemicalsphytochemicals

$

Distribution of rice bran fatty acid and total oil lipid content across a set of U.S. and exotic Oryza sativa germplasm grown in the same environment.

0

10

20

30

40

50

60

15.0 17.0 19.0 21.0 23.0 25.0 27.0 29.0

Oil content (% wt/wt)

Ran

cid

ity

(mg

C8:

0/g

)

r= -0.01

r = - 0.01

0

10

20

30

40

50

60

0.0 5.0 10.0 15.0 20.0 25.0

Esterase activity (mg C8:0/g)

Ran

cidi

ty (m

g C

8:0/

g)r = 0.89

0

10

20

30

40

50

60

15.0 17.0 19.0 21.0 23.0 25.0 27.0 29.0

Oil content (% wt/wt)

Ran

cid

ity

(mg

C8:

0/g

)

A1

A2

CB

0

5

10

15

20

25

30

35

40

45

9-11 11-13 13-15 15-17 17-19 19-21 21-23 23-25 25-27

Fe Content (ug /g)

Nu

mb

er o

f Lin

es

Parent #1Parent #2

Tocopherols

Tocotrienols

Vitamin E Complex • 8 homologs: , , ,

• Found in plant oils

• Rice bran oil and palm are rich sources of tocotrienols

• Tocotrienols inhabit cholesterol synthesis in laboratory animals

• Lower serum total and LDL cholesterol in humans

Gamma Oryzanol Fraction(ferulate esters of triterpene alcohol)

• Plant sterols (phytosterols)

• Structure similar to cholesterol

• 10 Compounds

• Health Effects

– UV protectant

– Anti-inflammatory

– Cholesterol lowering

020406080

240 290 340 390 440

Tocols (ug/g)

Freq

uenc

y

0

10

20

30

40

3.0 3.8 4.3 4.8 5.3 5.8 6.3

Gamma-Oryzanol (mg/g)

Freq

uenc

y

Distribution of rice bran tocol and gamma-oryzanol contents across a set of U.S. and exotic Oryza sativa germplasm grown in the same environment.

PhenolicsPhenolics

Phenolics are one of the most diverse groups of substances in the plant kingdom (> 8,000 compounds)

They are products of plant secondary metabolism, arising from the shikimate pathway

Their chemical structure ranges from simple compounds to highly polymerized substances like tannins

Phenolic Compounds: PropertiesPhenolic Compounds: Properties

Chemical PropertiesChemical Properties

Natural antioxidants Radical scavengers Metal chelators

Nutrition and Health PropertiesNutrition and Health Properties

Prevention of cancer and cardiovasculardiseases

Antimicrobial, anti-diarrheal, anti-viral, and anti-inflammatory effects Food sensory quality: astringency and

bitterness Some are anti-nutritive, i.e. tannins

Ecological PropertiesEcological Properties

Resistance to pathogenesand predators

UV-B tolerance Pigmentation Allelopathic effects Antifungical and antibacterial effects

Industrial ApplicationsIndustrial Applications

Food additives: natural colorants andpreservatives

Production of paints, paper, cosmetics Tanning agents

Plant Phenolics: ClassificationPlant Phenolics: ClassificationClassClass Basic skeletonBasic skeleton ExamplesExamples

MonophenolsMonophenols

Simple phenolsSimple phenols CC66 Resorcinol, thymolResorcinol, thymol

Phenolic acidsPhenolic acids CC6-6-CC11 Salicylic acid, Salicylic acid, pp-hydroxybenzoic acid-hydroxybenzoic acid

Cinnamic acidsCinnamic acids CC6-6-CC33 Caffeic acid, ferulic acidCaffeic acid, ferulic acid

PolyphenolsPolyphenols

FlavonoidsFlavonoids CC6-6-CC3-3-CC66 Flavones: isovitexin, luteolinFlavones: isovitexin, luteolin

Flavonols: quercetin, kaempferolFlavonols: quercetin, kaempferol

Anthocyanins: cyanidin glycosidesAnthocyanins: cyanidin glycosides

Flavanols: (+)-catechin, (-)-epicatechinFlavanols: (+)-catechin, (-)-epicatechin

Tannins Tannins (C(C6-6-CC3-3-CC66))nn Condensed tannins or proanthocyanidins:Condensed tannins or proanthocyanidins:

Oligomers and polymers of flavanolsOligomers and polymers of flavanols

Phenolic Content in Rice FractionsPhenolic Content in Rice Fractions

Total phenolic contentTotal phenolic content (mg PhlE/100 g)(mg PhlE/100 g)††

Brown RiceBrown Rice 27.6 27.6

Milled RiceMilled Rice 14.8 14.8

HuskHusk 128.4128.4

BranBran 272.2272.2

Source: Singaravadivel and Raj, 1979Source: Singaravadivel and Raj, 1979† Phloroglucinol equivalents per 100 g (dry weight basis)Phloroglucinol equivalents per 100 g (dry weight basis)

Light-Brown

Brown

Purple

Red

Per

cen

t (%

)

GAE/g whole grain*

0

20

40

60

80

100

120

140

0.0 20.0 40.0 60.0 80.0

Total phenolics (mg GA eq./g rice bran)

% o

f DP

PH

dep

lete

d a

rea

r2=0.994.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

3.0 3.5 4.0 4.5 5.0 5.5 6.0

Total phenolics (mg GA equiv./g DW rice bran)

% o

f D

PP

H d

ep

lete

d a

rea

r2= 0.67

† Purple rice bran

Red rice bran

Brown rice bran

Light-brown rice bran

‡ Dried fruits

Common berries

Whole grain wheat cereal/raisins

Whole grain wheat cereal

Whole grain oat cereal

100% whole grain bread

Corn cereal

Rice cereal

Common fruits

White bread

Common vegetables

Melons

Antioxidant Activity of Foods versus Rice Bran Antioxidant Activity of Foods versus Rice Bran

† Our data (, n = 17), with error bars; ‡ Source: Miller et al 2000 () * TE/100 g, µM trolox equivalents per 100 gram; Antioxidative activity measured using DPPH

0 5000 10000 15000 20000 25000 30000 35000 40000 45000

TE/100gTE/100g**

CultivarCultivar ColorColor FractionFraction TE/gTE/g‡‡

Dixiebelle light-brown low-P† 29tannins 148

Jacinto light-brown low-P 46tannins 201

EAS 3 red low-P 47tannins 2731

Paddy Vanhali red low-P 48tannins 2777

Pai Chiao pai red low-P 51tannins 2674

IAC black purple low-P 825tannins 1467

HB-1 purple low-P 79tannins 669

Antiradical Effect of Isolated Fractions Antiradical Effect of Isolated Fractions

† Low-P, low-molecular phenolics‡ ‡ TE/g, trolox equivalents in µM trolox per g (dry weight basis)

HPLC Analysis of Phenolic Standards

HPLC Analysis of Low-Molecular Phenolics from EAS 3 (Red Bran)HPLC Analysis of Low-Molecular Phenolics from EAS 3 (Red Bran)

Non-identified compounds

280 nm350 nm

280 nm350 nm

AU

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00

Polymeric Phenolics

Simple Phenolics

A

B

HPLC-RP Separations of Rice Bran Ethyl Acetate Extracts. A: ‘EAS-3’ (Peru). B: ‘Cypress’ (U.S.).

HPLC-RP Separations of Rice Bran Ethyl Acetate Extracts. A: ‘EAS-3’ (Peru). B: ‘Cypress’ (U.S.).

A

B

Future Collaborative Future Collaborative ResearchResearch

Fractionate individual phenolic Fractionate individual phenolic compoundscompounds

Identify their structures using MSIdentify their structures using MSUse preparative HPLC to collect Use preparative HPLC to collect

large quantities of the fractionslarge quantities of the fractionsEvaluate their individual radical Evaluate their individual radical

scavenging abilities and anti-cancer scavenging abilities and anti-cancer propertiesproperties

Evaluate ability to prevent rice bran Evaluate ability to prevent rice bran oil rancidityoil rancidity

Future Collaborative Future Collaborative ResearchResearch

Reducing rice bran rancidityReducing rice bran rancidityCross low esterase activity rice x Cross low esterase activity rice x

High yielding U.S. cultivarHigh yielding U.S. cultivar Use anti-sense technology to knock Use anti-sense technology to knock

out lipase and lipoxygenase activityout lipase and lipoxygenase activitySensory evaluation of productsSensory evaluation of products

Gene Expression

• Rice bran phytochemicals

• Are they modulating gene expression rates

• Microarray technology allows the evaluation of the expression of many genes from a genome at the same time

• Tocotrienol effects on enzyme activity – gene x nutrient interaction?

Goal = Improve the Goal = Improve the Nutritional Content of RiceNutritional Content of Rice

High phytochemical High phytochemical xx Low rancidity Low rancidity cultivar cultivar xx High Yielding cultivar High Yielding cultivar

High Yielding, Low rancidity, High Phytochemical