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
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.
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)
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