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Making Sense of Science
Megan Tichy, Ph.D.Lecturer, Texas A&M University
Toxic grains Scientific and non-scientific names
Quantification Parts per million
Gluten Chemical structure
Processing Distillation
Highlights
• Classification based on physical, behavioral or genetic diversity• Subject to revisions
Scientific names
Human Pea Wheat
Domain Eukarya Eukarya Eukarya
Kingdom Animalia Plantae Plantae
Phylum Chordata Magnoliophyta Magnoliophyta
Class Mammalia Magnoliopsida Lilliopsida
Order Primates Fabales Cyperales
Family Hominidae Fabaceae Poaceae
Genus Homo Pisum Triticum
Species H. sapiens P. sativum T. aestivum
More
sp
ecifi
c
Mnemonic device: Kids Prefer Cheese Over Fried Green Spinach
Poaceae
Festucoideae
Oryzeae - rice
Triticeae - wheat, rye, barley
Aveneae - oats
Panicoideae Andropogoneae - sorghum
Tripsaceae - corn
Paniceae - millet
KINGDOM – Plantae – plantsPHYLUM – Magnoliophyta – flowering plantsCLASS – Liliopsida – monocotyledonsORDER – Cyperales – grasses/sedges
The grass family
FAMILY
SUBFAMILY
TRIBE
Donald D. Kasarda, Ph.D., Research Chemist (Retired), USDA http://wheat.pw.usda.gov/ggpages/index.shtml
WheatTriticum aestivum
BarleyHordeum vulgare
RyeSecale cereale
Images: Home-Grown Cereals Authority (HGCA); http://www.hgca.com
Toxic grains
Durum
Hard Red Winter
Hard Red Spring
Hard White Wheat
Soft Red Winter
Soft White Wheat
Modern wheat varieties
Atta whole wheat, stone-grinded Bulgur whole wheat, precooked, dried, cracked, and sifted Couscous moistened semolina, rolled and shaped,
coated with finely ground wheat four Farina endosperm milled to fine granular consistency, sifted Seitan washed dough; water removes starch Semolina inner endosperm of wheat not ground into flour
Images: Wikipedia commons
Seitan (Taiwan)
Wheat products
Atta (India) Farina
Ancient forms of wheat
Einkorn Triticum monococcum DiploidEmmer (farro) Triticum dicoccum TetraploidSpelt (dinkel) Triticum spelta Hexaploid
Kamut Triticum turanicum Tetraploid
Genetics: Diploid = 14 chromosomesTetraploid = 28 chromosomesHexaploid = 42 chromosomes
More chromosomes = easier to breed varieties suitable for non-ideal soils/climates
Wheat proteins
Protein Soluble in
Albumin WATER - neutral pH
Globulin WATER - salt solutions
Prolamin ETHANOL - 70-90% alcohol
Glutelin NONE OF THE ABOVEdilute acids/bases, detergents, or reducing agents
Gluten
Cause of most wheat allergies
“It is highly probable that glutenin proteins are toxic. Attempts to breed wheat with satisfactory baking properties tolerated by coeliac patients will be very difficult.” Eur J Gastroenterol Hepatol. 2006 May;18(5):483-91.
Gluten proteins
GLUTEN
GLIADINS
GLUTENINS
HMW LMW
All coded by different genes (DNA)
Prolamine = proline + glutamine
Grain Prolamin fraction % total protein
Wheat Gliadin 69
Rye Secalin 30-50
Barley Hordein 46-52
Oats Avenin 16
Millet Panicin 40
Corn Zien 55
Rice Orzenin 5
Sorghum Kafirin 52
Grain proteins
toxic safe still questionedcontaminated? pure?
BranRich source of magnesium and iron. ¼ cup contains 6 grams fiber.
GermEmbryo
vitamin E, magnesium, riboflavin, thiamin,
phosphorus, niacin, iron and zinc. Some fat and protein.
Manufacturers remove the germ and bran to make refined bread flour
Anatomy of a kernel
EndospermStorage tissuestarch, gluten
Whole-grain: endosperm, bran, and embryo (all three parts of the kernel!) left intact
flour
dough
starch (sugars)75%
gluten (protein)12% (varies)
Wheat flour: gluten and starch
H2O
C6H12O6 + 6 O2 → 6 CO2 + 6 H2O + energy
glucose + oxygen → carbon dioxide + water
doughyeast bread
Image: http://www.exploratorium.edu/cooking/bread/activity-gluten.html
• Flour contains 10-15% protein• The protein is 80% gluten
Q: If 1 slice of bread uses 40 g flour how much gluten is in it?A: solve for protein 40 g X 0.13 = 5.2 g protein solve for gluten 5.2 g X 0.8 = 4 g gluten
Q: How many mg gluten is in 1/8 teaspoon flour?A: Conversion for wheat flour 1 tsp = 2.5 g 1/8 tsp all purpose flour = 0.31 g flour 0.31 g X 0.13 X 0.8 = 0.033 g gluten 1/8 tsp 33 mg gluten
How much gluten?
Measuring spoon image: http://www.tubularspices.com/accessories/spoons.asp
How much is too much?
Codex Alimentarius (WHO/FAO), has two limits for “gluten-free”: 0.02% gluten content - rendered gluten-free0.002% gluten content - naturally gluten-free food
Normal gluten intake by healthy individuals = 13 g gluten per dayCompare – 50 mg (0.05 g) vs. 13 gNormal intake is 260 times as much as the suggested threshold
“Ingestion of contaminating gluten should be kept lower than 50 mg/day in the treatment of CD” – C. Catassi et al. AmJ Clin Nutr 2007;85:160–6. Some people are far more sensitive*
A person could ingest 3-6 mg gluten per day by consuming 6-8 ounces of “naturally gluten free” grain food
*Reference: A milligram gluten a day keeps the mucosal recovery away: a case report, F. Biagi, J. Campanella and S. Martucci et al., Nutr Rev 62 (2004), pp. 360–363
0.002% = 20 parts per million or ppm
A part per what?
Examples of a part per twelve• One egg out of a dozen• One cupcake out of a dozen• One pane from this stained-glass window
per cent means one part per hundred X 100 =
A part per hundred (%)
One bushel of wheat (60 lbs) contains 1 million individual kernels
A part per million (ppm)
A part per million (ppm)
Use % for larger fractions 1 part per 100 = 0.01 = 1%
Use ppm for smaller fractions 1 part per 1,000,000 = 0.000001 = 0.0001%
Calculating ppm:• Start with the fraction (part/whole)• Rather than 2 decimal places to the right → %• Move 6 decimal places to the right → ppm
20 ppm = 20 minutes in:A) 4 days
B) 10 weeks C) 2 years
20 minutes
4 days
1 hour
60 minutes
1 day
24 hours20
5760
= 0.003472= 3,472 ppm
=
20 minutes
2 years
1 hour
60 minutes
1 day
24 hours20
10512001 year
365 days
= 0.00001902= 19 ppm
=
Quiz answer
20 ppm
20 seconds in 11.5 days
20 inches in 15.8 miles
20 ounces in 62,500 pounds
20¢ in $10,000
Perspective: How much is 20 ppm?
Tiny amounts are biologically significant!Most fish need 4 ppm dissolved oxygen to survive; Bass and trout seek out waters with 9-12 ppm oxygen
Using ppm to calculate quantity gluten
ppm gluten
2 g
Tsp non-dairy GF creamer
20 g
10 GF crackers
200 g
4 slices GF bread
20 0.04 mg 0.4 mg 4 mg
50 0.1 mg 1 mg 10 mg
100 0.2 mg 2 mg 20 mg
200 0.4 mg 4 mg 40 mg
Reminder: The ingestion of contaminating gluten should be kept lower than 50 mg/day in the treatment of CD
Proteins
Contain carbon, oxygen, hydrogen, nitrogen, often sulfur
Broken down to 22 naturally occurring amino acids
Starch vs. Protein
Starches
Contain carbon, oxygen, hydrogen
Broken down to only one material, glucose
HN
NH
HN
O
O
O
NH
HN
HN
HO
O
O
O
NH
O
OH SH
OH
SH
O
HOOH
OHO
HOHO
OOH
OH
O
HOOH
OHO
OHO
OOH
OH
O
glucose
aminoacid
I Have Received Much Kudos For Learning These Very Well
Mnemonic device:
O
OHH2N
IsoleucineIleI
HistidineHisH
OHO
NH2
HN
N
ArginineArgR
O
OHH2N
HN
HN
NH2 MethionineMetM
O
OHH2N
S
LysineLysK
O
OHH2NH2N
PhenylalaninePheF
O
OHH2N
LeucineLeuL
O
OHH2N
ThreonineThrT
O
OHH2N
OH
ValineValV
O
OHH2N
TryptophanTrpW
O
OHH2N
NH
Essential amino acids (from diet)
All amino acids have a 1-letter abbreviation
Proteins are polymers of amino acids with directionalityC (carboxy)-terminus → N (amino)-terminus
“Beads on a string”
Protein primary structure
Stability: Proteins vary widely in their stability to heatProline is known to provide stability to proteins
MKTFLILALLAIVATTATTAVRVPVPQLQPQNPSQQQPQ
EQVPLVQQQQFLGQQQPFPPQQPYPQPQPFPSQQPYLQL
QPFLQPQLPYSQPQPFRPQQPYPQPQPQYSQPQQPISQQQ
QQQQQQQQQQQQQQQQIIQQILQQQLIPCMDVVLQQHN
IVHGKSQVLQQSTYQLLQELCCQHLWQIPEQSQCQAIHN
VVHAIILHQQQKQQQQPSSQVSFQQPLQQYPLGQGSFRP
SQQNPQAQGSVQPQQLPQFEEIRNLARK
Gliadin primary structure (sequence)
Q: Why does gliadin survive in the presence of enzymes and heat?
MKTFLILALLAIVATTATTAVRVPVPQLQPQNPSQQQPQ
EQVPLVQQQQFLGQQQPFPPQQPYPQPQPFPSQQPYLQL
QPFLQPQLPYSQPQPFRPQQPYPQPQPQYSQPQQPISQQQ
QQQQQQQQQQQQQQQQIIQQILQQQLIPCMDVVLQQHN
IVHGKSQVLQQSTYQLLQELCCQHLWQIPEQSQCQAIHN
VVHAIILHQQQKQQQQPSSQVSFQQPLQQYPLGQGSFRP
SQQNPQAQGSVQPQQLPQFEEIRNLARK
Reference: L. Shan, O. Molberg and I. Parrot et al., Structural basis for gluten intolerance in celiac sprue, Science 297 (2002), pp. 2275–2279.
Secondary structure: Regularly repeating local structures stabilized by hydrogen bonds. Ex: alpha helix, beta sheet
Tertiary structure: Overall shape or “fold”; the spatial relationship of the secondary structures relative to one another
Quaternary structure: Results from interaction of more than one protein molecule (subunit) which functions as part of the larger assembly or protein complex.
Protein folding
amino acids
binding site
Protein function requires folding
•Structure: keratin •Transport: hemoglobin•Storage: myoglobin•Hormones: insulin •Catalysis: lactase
5 common protein functions:
Just a few “beads” from the string ≠ gluten
The average person consumes• 10 - 20 g bound glutamate/day• 0.5 - 1.5 g MSG/day
MSG ≠ gluten
Na+
NH2
O
-O
O
OH
monosodium glutamate(both L and D)
NH2
O
H2N
O
OH
L-glutamine
NH2
O
HO
O
OH
L-glutamic acid
L versus D• In higher mammals, all proteins contain “L” amino acids. • During hydrolysis “L” → “D” • The “D” form is tasteless
Other small molecules ≠ gluten
O
N
N
NN
O The structure of caffeine
Not all symptoms are related to celiac disease
Calcium oxalate can form crystals which eventually grow into kidney stones
Oxalate binds with calcium in your urinary tract
Ca2+OO-
O-
O
The structure of calcium oxalate
200-300 milligrams daily appears to be safe
Starbucks’ Tall Coffee = 260 milligrams
How do we test for gluten?ELISA – Enzyme Linked Immunosorbent AssayELISA detection limit between 1-2 ppm, with <0.5 ppm reported*
Any of the following can be made from wheat starch:• Maltodextrin, glucose syrup, dextrose• Poly-ols, sugar alcohols (sorbitol, xylitol, mannitol etc…)• Lactic acid, citric acid, acetic acid (vinegar), ascorbic acid
Hydrolyzed wheat starch
*Sousa et al. Am J Clin Nut, Vol. 87, No. 2, 405-414, February 2008
North America – starch products usually derived from corn
Hydrolysis = hydro (water) + lysis (cleavage/breakage) A chemical reaction in water that breaks down wheat starch
Alcoholic fermentation
A vessel allows carbon dioxide to escape, but prevents outside air from coming in
C6H12O6 → 2 C2H5OH + 2 CO2
glucose → ethanol + carbon dioxide
Without oxygen present:
Wine/brandy - natural sugars present in fruits Rum - cane sugar or molasses Whiskey - amylase-treated grain Vodka - amylase-treated grain or potatoes Gin - grain infused with juniper berry
Safe liquors
Distillation: separation based on volatility
Volatile
Non-volatile
Volatility - how readily substances become gasesStronger intermolecular forces decrease volatilityIt takes more energy to break these molecules apart
NaCl
H3N CH C
CH2
O
O
CH2
C
NH2
O
glutamine
H2C
CH3HO
ethanol
CCH3
O
O
H2C
H3C
ethyl acetateH3C
C
O
CH3
acetone
H2O water
Also not volatile:large peptides (proteins)starches, any polymer
Not solely based on sizeof the molecule
Ethyl acetateMolecular Weight: 88 g/moleBoiling Point: 77 C C
CH3
O
O
H2C
H3C
H3C
C
O
CH3
EthanolMolecular Weight: 32.04 g/moleBoiling Point: 64.7 C
AcetoneMolecular Weight: 58.08 g/mole Boiling Point: 56.5 C
ButanolMolecular Weight: 74.12 g/moleBoiling point: 117.7 C
H2C
CH3HO
H2C
OHCH2
H2C
H3C
Incr
easi
ng b
oili
ng p
oin
tVolatility ≠ size
3 C’s1 O
2 C’s1 O
4 C’s1 O
4 C’s1 O
Simple distillation procedure
The separated liquid (distillate) drips out into a new chamber
Volatile portion enters gas phase Tubes are cooled
gas → liquid
Ethanol gas floats upcirculates throughcondensing tubes
Liquid heated in boiling chamber
Peptides/amino acidsdo not evaporate
Conclusions
There are MANY other names for wheat Spelt is NOT an alternative for wheat!
A ppm is not a quantity 20 ppm can be many different quantities, depends on how much you ate!
Gluten is a highly stable protein fraction Several different fragments of gliadin and glutenin are toxic Small molecules do not mimic gluten, but can have negative health effects of their own
Distillation is separation based on volatility Distilled products are gluten-free as long as gluten was not added post-distillation
THANK YOU!
Brazos Valley Gluten Intolerance GroupDenise Fries, Taylor Jensen Julie Hoyle (secretary), JenSara Boswell Kim Melissari (treasurer), LaurenMonica Michalka
FamilyShane (husband), Nathan (son)Karen Jones & FamilyMcLean family – Rochester, NYSantiago family – Bryan, TX
GIG mentorsCynthia Kupper, RDBetty Barfield
Texas A&M UniversityDr. Ganesa Gopalakrishnan